5′-triphosphate oligoribonucleotides

ABSTRACT

Disclosed herein are synthetic oligoribonucleotides that form hairpin loop structures. The oligoribonucleotides can be used in the treatment of viral infection including prophylactic treatments. The oligoribonucleotides can also be used as adjuvants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/802,187 filed Jul. 17, 2015, which claims priority to U.S.Provisional Patent Application No. 62/026,473, filed Jul. 18, 2014. Thisapplication is also related to U.S. patent application Ser. No.14/177,866 filed Feb. 11, 2014. Each of these referenced applications isincorporated herein by reference in its entirety as if fully set forthherein.

FIELD

Generally, the field is RNA-based therapeutic molecules. Morespecifically, the field is 5′-triphoshpate oligoribonucleotide immunesystem agonists and pharmaceutical compositions comprising the same.

BACKGROUND

The innate immune system has evolved numerous molecular sensors andsignaling pathways to detect, contain and clear viral infections(Takeuchi O and Akira S Immunol Rev 227, 75-86 (2009); Yoneyama M andFujita T, Rev Med Virol 20, 4-22 (2010); Wilkins C and Gale M Curr OpinImmunol 22, 41-47 (2010); and Brennan K and Bowie A G Curr OpinMicrobiol 13, 503-507 (2010); all of which are incorporated by referenceherein.) Viruses are sensed by a subset of pattern recognition receptors(PRRs) that recognize evolutionarily conserved structures known aspathogen-associated molecular patterns (PAMPs). Classically, viralnucleic acids are the predominant PAMPs detected by these receptorsduring infection. These sensing steps contribute to the activation ofsignaling cascades that culminate in the early production of antiviraleffector molecules, cytokines and chemokines responsible for theinhibition of viral replication and the induction of adaptive immuneresponses (Takeuchi O and Akira S Cell 140, 805-820 (2010), Liu S Y etal, Curr Opin Immunol 23, 57-64 (2011); and Akira S et al, Cell 124,783-801 (2006); all of which are incorporated by reference herein). Inaddition to the nucleic acid sensing by a subset of endosome-associatedToll-like receptors (TLR), viral RNA structures within the cytoplasm arerecognized by members of the retinoic acid-inducible gene-I (RIG-I)-likereceptors (RLRs) family, including the three DExD/H box RNA helicasesRIG-I, Mda5 and LGP-2 (Kumar H et al, Int Rev Immunol 30, 16-34 (2011);Loo Y M and Gale M, Immunity 34, 680-692 (2011); Belgnaoui S M et al,Curr Opin Immunol 23, 564-572 (2011); Beutler B E, Blood 113, 1399-1407(2009); Kawai T and Akira S, Immunity 34, 637-650 (2011); all of whichare incorporated by reference herein.)

RIG-I is a cytosolic multidomain protein that detects viral RNA throughits helicase domain (Jiang F et al, Nature 479, 423-427 (2011) andYoneyama M and Fujita T, J Biol Chem 282, 15315-15318 (2007); both ofwhich are incorporated by reference herein). In addition to its RNAsensing domain, RIG-I also possesses an effector caspase activation andrecruitment domain (CARD) that interacts with the mitochondrial adaptorMAVS, also known as VISA, IPS-1, and Cardif (Kawai T et al, Nat Immunol6, 981-988 (2005) and Meylan E et al, Nature 437, 1167-1172 (2005), bothof which are incorporated by reference herein.) Viral RNA binding altersRIG-I conformation from an auto-inhibitory state to an open conformationexposing the CARD domain, resulting in RIG-I activation which ischaracterized by ATP hydrolysis and ATP-driven translocation of RNA(Schlee M et al, Immunity 31, 25-34 (2009); Kowlinski E et al, Cell 147,423-435 (2011); and Myong S et al, Science 323, 1070-1074 (2011); all ofwhich are incorporated by reference herein). Activation of RIG-I alsoallows ubiquitination and/or binding to polyubiquitin. In recentstudies, polyubiquitin binding has been shown to induce the formation ofRIG-I tetramers that activate downstream signaling by inducing theformation of prion-like fibrils comprising the MAVS adaptor (Jiang X etal, Immunity 36, 959-973 (2012); incorporated by reference herein). MAVSthen triggers the activation of IRF3, IRF7 and NF-κB through theIKK-related serine kinases TBK1 and IKKε (Sharma S et al, Science 300,1148-1151 (2003); Xu L G et al, Molecular Cell 19, 727-740 (2005); andSeth R B et al, Cell 122, 669-682 (2005); all of which are incorporatedby reference herein). This in turn leads to the expression of type Iinterferons (IFNβ and IFNα), as well as pro-inflammatory cytokines andanti-viral factors (Tamassia N et al, J Immunol 181, 6563-6573 (2008)and Kawai T and Akira S, Ann NY Acad Sci 1143, 1-20 (2008); both ofwhich are incorporated by reference herein.) A secondary responseinvolving the induction of IFN stimulated genes (ISGs) is induced by thebinding of IFN to its cognate receptor (IFNα/βR). This triggers theJAK-STAT pathway to amplify the antiviral immune response (Wang B X andFish E N Trends Immunol 33, 190-197 (2012); Nakhaei P et al, Activationof Interferon Gene Expression Through Toll-like Receptor-dependent and-independent Pathways, in The Interferons, Wiley-VCH Verlag GmbH and CoKGaA, Weinheim F R G (2006); Sadler A J and Wiliams B R, Nat Rev Immunol8, 559-568 (2008); and Schoggins J W et al, Nature 472, 481-485 (2011);all of which are incorporated by reference herein.)

The nature of the ligand recognized by RIG-I has been the subject ofintense study given that PAMPs are the initial triggers of the antiviralimmune response. In vitro synthesized RNA carrying an exposed 5′terminal triphosphate (5′ppp) moiety was identified as a RIG-I agonist(Hornung V et al, Science 314, 994-997 (2006); Pichlmair A et al,Science 314, 997-1001 (2006); and Kim D H et al, Nat Biotechol 22,321-325 (2004); all of which are incorporated by reference herein). The5′ppp moiety is added to the end of all viral and eukaryotic RNAmolecules generated by RNA polymerization. However, in eukaryotic cells,RNA processing in the nucleus cleaves the 5′ppp end and the RNA iscapped prior to release into the cytoplasm. The eukaryotic immune systemevolved the ability to distinguish viral ‘non-self’ 5′ppp RNA fromcellular ‘self’ RNA through RIG-I (Fujita T, Immunity 31, 4-5 (2009);incorporated by reference herein). Further characterization of RIG-Iligand structure indicated that blunt base pairing at the 5′ end of theRNA and a minimum double strand (ds) length of 20 nucleotides were alsoimportant for RIG-I signaling (Schlee M and G Hartmann, MolecularTherapy 18, 1254-1262 (2010); incorporated by reference herein). Furtherstudies indicated that a dsRNA length of less than 300 base pairs led toRIG-I activation but a dsRNA length of more than 2000 bp lacking a 5′ppp(as is the case with poly I:C) failed to activate RIG-I. (Kato H et al,J Exp Med 205, 1601-1610 (2008); incorporated by reference herein).

RNA extracted from virally infected cells, specifically viral RNAgenomes or viral replicative intermediates, was also shown to activateRIG-I (Baum A et al, Proc Natl Acad Sci USA 107, 16303-16308 (2010);Rehwinkel J and Sousa C R E, Science 327, 284-286 (2010); and RehwinkelJ et al, Cell 140, 397-408 (2010); all of which are incorporated byreference herein). Interestingly, the highly conserved 5′ and 3′untranslated regions (UTRs) of negative single strand RNA virus genomesdisplay high base pair complementarity and the panhandle structuretheoretically formed by the viral genome meets the requirements forRIG-I recognition. The elucidation of the crystal structure of RIG-Ihighlighted the molecular interactions between RIG-I and 5′ppp-dsRNA(Cui S et al, Molecular Cell 29, 169-179 (2008); incorporated byreference herein), providing a structural basis for the conformationalchanges involved in exposing the CARD domain for effective downstreamsignaling.

SUMMARY

Disclosed herein is a synthetic oligoribonucleotide at least 41nucleotides in length that can form a hairpin structure comprising atleast 17 base pairs. The synthetic oligoribonucleotide further comprisesa triphosphate group at its 5′ end. Examples of the oligoribonucleotidecan include sequences such as SEQ ID NO: 10, SEQ ID NO: 11, or SEQ IDNO: 12 described herein. The oligoribonucleotide can also be of at least99 nucleotides in length and can form a hairpin structure of at least 48base pairs. Examples of this aspect of the oligonucleotide can includesequences such as SEQ ID NO: 15 or SEQ ID NO: 16 described herein. Insuch an oligoribonucleotide, the hairpin structure can comprise at least26 consecutive U-A base pairs. Examples of this aspect of theoligoribonucleotide can include sequences such as SEQ ID NO: 13, SEQ IDNO: 14 and SEQ ID NO: 17 described herein.

Further disclosed are pharmaceutical compositions comprising atherapeutically effective amount of any of the disclosed syntheticoligoribonucleotides and a pharmaceutically acceptable carrier. Suchcompositions can also include viral antigens such as an influenza viruslike particle and be formulated as a vaccine.

Further disclosed are methods of treating a viral infection such asvesicular stomatitis virus, dengue virus, human immunodeficiency virus,chikungunya virus, or influenza virus.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some of the drawings were provided in color and can be better understoodthrough the use of color reproduction. Applicants consider the colordrawings to be part of the original disclosure and reserve the right toprovide color drawings in later proceedings.

FIG. 1: M8 elicits a more robust innate response compared to other RIG-Iagonists.

FIG. 1 is a set of four bar graphs summarizing the results when lungepithelial A549 cells were transfected with VSV WT, M5, M8, poly (I:C),or CL9 aptamer at the indicated concentrations.

Total RNA was extracted, subjected to reverse transcription, andanalyzed by real-time PCR using CXCL10-, IL1a-, IL29-, TNFα-, andGAPDH-specific primers.

FIG. 2A: In vitro transcription of the disclosed5′ppp-oligonribonucleotides produces a single RNA product. FIG. 2A is animage of a 15% TBE-urea polyacrylamide gel showing in vitro transcribedWT and selected mutants therein—M1, M2, M3, M4, and M5 afterpurification via spin column.

FIG. 2B: In vitro transcribed VSV WT mRNA 5′ppp-oligoribonucleotideproduct is sensitive to RNase. FIG. 2B is an image of a 15% TBE-ureapolyacrylamide gel showing synthesized and purified WT subjected toRNase A or DNase I treatment as indicated.

FIG. 2C: In vitro transcribed M5 product is sensitive to RNase. FIG. 2Cis an image of a 15% TBE-urea polyacrylamide gel showing synthesized andpurified M5 (5′pppSEQ ID NO: 10) subjected to RNase A or DNase Itreatment as indicated.

FIG. 3A: M5 is a 5′-triphosphate RIG-I agonist that induces more IFN-βthan the prototypical WT structure. FIG. 3A is a bar graph summarizingthe results of A549 cells transfected with reporter assay plasmids thentransfected with the indicated RNA agonists at the given concentrationsfor 24 hours. IFN-β reporter gene activity was then measured by theDual-Luciferase Reporter Assay kit.

FIG. 3B: M5 induces cytokines to a higher level than other RIG-Iagonists as well as other immunostimulants. FIG. 3B is a set of threebar graphs summarizing the results of A549 cells transfected with 0.1ng/ml WT, M5, poly (I:C), or CL2 aptamer for 24 hours. Total RNA wasextracted, subjected to reverse transcription, and analyzed by real-timePCR using ISG56-, IFNβ-, IL1a-, and GAPDH-specific primers.

FIG. 3C: M5 inhibits influenza replication in vitro. FIG. 3C is an imageof an immunoblot of A549 cells transfected with 0.01 ng/ml of each ofthe 5′pppRNA variants for 24 hours then infected or not with influenzaH1N1 strain A/PR/8/34 (MOI 0.2). After 24 hours of infection, whole cellextracts were resolved by native gel electrophoresis and revealed byimmunoblot using NS1, ISG56, pSTAT1, and β-actin antibodies.

FIG. 3D: Dengue viral RNA synthesis is completely inhibited inM5-treated cells. FIG. 3D is a bar graph summarizing the results whenA549 cells were transfected with 1 ng/ml of 5′pppRNAs including M5(5′ppp-SEQ ID NO: 10), aptamers, or poly (I:C), then treated with denguevirus serotype 2 strain NGC (MOI 0.5). Total RNA was extracted,subjected to reverse transcription, and analyzed by real-time PCR usingdengue- and GAPDH-specific primers.

FIG. 3E: M5 (comprising SEQ ID NO: 10) antiviral activity against dengueis superior to other RIG-I agonists and other immunostimulants. A549cells were transfected with WT, M5, poly (I:C), or CL2 aptamer at arange of concentrations for 24 hours then challenged with dengue virus(MOI 0.5) for 24 hours. Intracellular staining (ICS) and flow cytometrywas performed to quantify the percentage of dengue E protein-positivecells.

FIG. 4A: Increased dsRNA length provides enhanced antiviral activityagainst dengue virus. FIG. 4A is a bar graph summarizing the resultswhen A549 cells were transfected with M5, M6, M7, and M8 5′pppRNA(5′pppSEQ ID NO: 10, 5′pppSEQ ID NO: 11, 5′pppSEQ ID NO: 12, and5′pppSEQ ID NO: 13, respectively) at a range of concentrations (0.01,0.1, 1, and 10 ng/ml) for 18 hours then challenged with dengue virus(MOI 0.5) for 24 hours. ICS staining and flow cytometry was performed toquantify the percentage of dengue E protein-positive cells.

FIG. 4B: M8 completely inhibits dengue viral RNA in vitro. FIG. 4B is abar graph summarizing the results when A549 cells were transfected withM5, M6, M7, or M8 at the indicated concentrations (0.01, 0.1, 1, and 10ng/ml) for 18 hours then challenged with dengue virus (MOI 0.5) for 24hours. Total RNA was extracted, subjected to reverse transcription, andanalyzed by real-time PCR using dengue- and GAPDH-specific primers.

FIG. 4C: Minimal concentrations of M8 are required to completely blockviral infection. FIG. 4C is a bar graph summarizing the results whenA549 cells were transfected with M8 (5′pppSEQ ID NO: 13) at lowconcentrations (0.1 to 0.000046 ng/ml at 1:3 dilutions). ICS stainingand flow cytometry was performed to quantify the percentage of dengue Eprotein-positive cells.

FIG. 5: Comparison of WT, M5, and M8 5′pppRNA structures. FIG. 5 is adrawing of secondary structures of optimized oligonucleotides generatedusing the RNAfold Web Server (University of Vienna).

FIG. 6A: The antiviral activity of M8 works through RIG-I. FIG. 6A is abar graph summarizing the expression of RIG-I or TL3/MDA5 were inhibitedby siRNA in A549 cells for 48 hours. Cells were then treated with M8(0.1 ng/ml) for 24 hours, infected with dengue (MOI 0.5) and viralreplication was evaluated 24 hours later by ICS staining and flowcytometry to quantify the percentage of dengue E protein-positive cells.

FIG. 6B: M8 activity is dependent on the RIG-I. FIG. 6B is an image ofan immunoblot of the results when A549 cells in which expression ofRIG-I, TLR3, or MDA5 was knocked down, were treated or not (NT) with M8for 24 hours. Whole cell extracts were resolved by native gelelectrophoresis and revealed by immunoblot using RIG-I, TLR3, MDA5,STAT1, and β-actin antibodies.

FIG. 7A: Enhanced antiviral activity against influenza is evident in M8(comprising SEQ ID NO: 13)-treated A549 cells. FIG. 7A is an image of animmunoblot of the results when A549 cells were pre-treated with WT, M5,or M8 (10, 1, 0.1, and 0.01 ng/ml) for 24 hours, and then infectedinfluenza H1N1 strain A/PR/8/34 (MOI 0.2). After 24 hours of infection,whole cell extracts were resolved by native gel electrophoresis andrevealed by immunoblot using NS1, pSTAT1, ISG56, and β-actin antibodies.

FIG. 7B: M8 inhibits dengue virus infection in human primary immunecells. FIG. 7B is a bar graph summarizing the results whenmonocyte-differentiated dendritic cells (MDDCs) were transfected withWT, M5, or M8 at the indicated concentrations (1000, 100, 10, and 1ng/ml) for 24 hours and infected with dengue virus (MOI 10). After 24hours, intracellular dengue replication was evaluated by ICS stainingand flow cytometry to quantify the percentage of dengue Eprotein-positive cells.

FIG. 7C: M8 (comprising SEQ ID NO: 13) induces primary human dendriticcell maturation. MDDC were transfected with WT, M5, M8, poly (I:C), orLPS (1 ng/ml) for 24 hours. After 36 hours, CD83, CD86, and CCR7expression levels were evaluated by surface staining and flow cytometry.

FIG. 8A is a drawing of the structure of a sequence based on M8(M8A-5′pppSEQ ID NO: 14). M8A was designed by changing part of thesequence of M8. Like M8, M8A is 99 nucleotides in length.

FIG. 8B is a drawing of a second sequence based on M8 (M8B-5′pppSEQ IDNO: 15). M8B was designed by changing the entire sequence of M8. Like M8and M8A, M8B is 99 nucleotides in length. Secondary structures in bothFIG. 8A and FIG. 8B were generated with the RNAfold Web Server(University of Vienna).

FIG. 9A: M8-derived sequences inhibit DENV replication more effectivelythan modified forms of M8. FIG. 9A is a bar graph summarizing theresults when A549 cells were transfected with M8, M8A, or M8B atconcentrations from (0.000001 to 1 ng/ml) for 24 hours then infectedwith dengue virus (MOI 0.5). After 24 hours, intracellular denguereplication was evaluated by ICS staining and flow cytometry to quantifythe percentage of dengue E protein-positive cells.

FIG. 9B: M8 induces antiviral cytokines more effectively than modifiedforms of M8. FIG. 9B is a set of six bar graphs summarizing the resultswhen A549 cells were transfected with M8, M8A, or M8B (0.01 ng/ml) for24 hours. Total RNA was extracted, subjected to reverse transcription,and analyzed by real-time PCR using ISG56-, IFN-β-, IL-1a, IL6, CXCL10-,TNFα-, and GAPDH-specific primers.

FIG. 10: Vaccination Schedule. FIG. 10 is a timeline of mousevaccination experiments. Numbers above line indicate weeks.

FIG. 11A: Vaccination with a virus like peptide-M5 combination increasesHA-specific total IgG compared to VLP or M5 alone. FIG. 11A is a plotshowing total HA-specific IgG quantified in vaccinated mice two weeksafter the booster vaccine. X axes represents sample dilutions and Y axesrepresents absorbance at 414 nm.

FIG. 11B: Total IgG titers are enhanced in VLP-M5 treated mice. FIG. 11Bis a plot of total IgG titers in sera of vaccinated mice two weeks afterthe booster vaccine. Titer was determined by ELISA.

FIG. 11C: VLP-M5 vaccination induces higher neutralizing antibody titersagainst influenza. FIG. 11C is a plot of titers of HA neutralizingantibodies in sera of vaccinated mice. Titer was determined byhemagglutination inhibition assay (HAI).

FIG. 11D: VLP- and VLP+M5-treated mice are protected from lethalinfluenza virus challenge. FIG. 11D is a plot of percent weight change(Y axes) in vaccinated mice upon challenge with reassorted H5N1 overtime (days, X axes).

FIG. 12A: VLP-M5 vaccinated mice survive lethal challenge of influenzaA. FIG. 12A is a Kaplan-Meier survival function of vaccinated mice uponchallenge with reassorted H5N1. Green line indicates mice vaccinatedwith M5+VLP, dark red line indicates mice vaccinated with VLP only, andpurple line indicates mice vaccinated with M5 and control mice.

FIG. 12B: VLP-M5 vaccinated mice do not develop pathologic illness afterchallenge with H5N1. FIG. 12B is a plot of the relative sickness scoreof mice vaccinated according to the indicated conditions upon challengewith reassorted H5N1.

FIG. 12C: Influenza-induced cell death was inhibited in VLP and VLP-M5vaccinated animals. FIG. 12C is a plot of apoptosis observed by TUNELassay in lungs of vaccinated mice upon challenge with reassorted H5N1.

FIG. 13: Inflammation in bronchial airways of animal challenged withreassorted H5N1. FIG. 13 is a set of four photomicrographs showing theresults of histopathological examination of H&E stained lung sections inmice vaccinated under the indicated conditions upon challenge withreassorted H5N1. Arrow indicates presence or absence of inflammationaround bronchial tubes.

FIG. 14A: Intramuscular inoculation of M5 stimulates antiviral IFN-βmRNA in mouse skin. FIG. 14A is a plot summarizing an RT-PCR assessmentof IFN-β mRNA levels in the quadriceps muscles of mice injected withvarious amounts of M5 (comprising SEQ ID NO: 10) compared to control.

FIG. 14B: Intramuscular inoculation of M8 stimulates antiviral IFN-βmRNA in mouse skin. FIG. 14B is a plot summarizing an RT-PCR assessmentof IFN-β mRNA levels in the quadriceps muscles of mice injected withvarious amounts of M8 compared to control.

FIG. 15A: M5 and M8 prevent foot swelling associated with chikungunyainfection.

FIG. 15A is a plot summarizing the results when mice were inoculatedintravenously with 2 or 10 μg of control, WT, M5 or M8 with in vivoJetPEI then injected with chikungunya virus via the footpad. Ipsilateralfoot swelling of mice was measured with a caliper over ten days.

FIG. 15B: Chikungunya viremia is reduced in M5 and M8 treated mice. FIG.15B is a plot summarizing the results when mice were injected with 2 or10 μg of control, WT, M5 or M8 along with in vivo JetPEI, then injectedwith chikungunya virus via the footpad. At 48 hours post-infection, RNAfrom the leg muscle was extracted and the number of genome copies ofchikungunya RNA was measured by real time qPCR.

FIG. 16A is a schematic representation of 5′pppRNA sequences thatinclude variations of the wild type (WT) VSV-derived 5′pppRNA (M1-M8),SELEX-selected RIG-I aptamers, and poly (I:C).

FIG. 16B is an image of a gel showing in vitro transcribed 5′pppRNA thatwas DNase-treated, purified and then run on a denaturing TBE-urea gel.

FIG. 16C is a set of bar graphs showing the results from A549 cells thatwere transfected with WT, M5, M8, CL9 aptamer, or poly (I:C) (2 fmol)using Lipofectamine RNAiMax. After 24 h, cells were harvested and totalmRNA was isolated. Antiviral and inflammatory gene expression wasdetermined by qPCR. Data are from two independent experiments performedin triplicate and represent the means±SEM.

FIG. 16D is a set of bar graphs showing the results from HEK293T cellsthat were co-transfected ISRE or IFN-β promoter reporter plasmid (200ng) along with WT, M5, or M8 5′pppRNA (10 ng/ml). Luciferase activitywas analyzed 24 h post-transfection by the Dual-Luciferase Reporterassay. Relative luciferase activity was measured as fold inductionrelative to the basal level of reporter gene. Data are from twoindependent experiments performed in triplicate and represent themeans±SEM.

FIG. 16E is an image of an immunoblot showing the results from A549cells that were transfected with 5′pppRNA (0.1, 1, or 10 ng/ml) andwhole cell extracts were prepared, resolved by SDS-PAGE, and analyzed byimmunoblotting for IRF3 pSer396, IRF3, ISG56, STAT1, RIG-I, and β-actin24 h later. One representative Western blot out of three independenttriplicates is shown.

FIG. 16F is an image of an immunoblot showing the results from A549cells that were transfected with 5′pppRNA or CL9 aptamer (0.01, 0.1, 1,or 10 ng/ml) for 24 h then infected with influenza (MOI 0.2) for 24 h.Whole cell extracts were prepared, resolved by SDS-PAGE, and analyzed byimmunoblotting for influenza viral protein NS1 and β-actin. Onerepresentative Western blot from one experiment is shown.

FIG. 16G is a bar graph showing the results from A549 cells that weretransfected with WT, M5, or M8 5′pppRNA, poly (I:C), or CL9 aptamer (1ng/ml) for 24 h then challenged with dengue virus (MOI 0.5). Percentageof infected cells was determined 24 h post-infection by intracellularstaining of DENV E protein expression. Data are from two independentexperiments performed in triplicate and represent the means±SEM.

FIG. 17A is a schematic representation of modifications to the M85′pppRNA. Sequence changes were made to the poly AU base-pair stretch(MBA, M8C), the WT-derived blunt-end (M8D), and the entire sequence(M8B) while keeping the structure intact.

FIG. 17B is a set of bar graphs showing the results of A549 cells thatwere transfected with 5′pppRNA (1 ng/ml) using Lipofectamine RNAiMax®.After 24 h, cells were harvested and total mRNA was isolated. Antiviraland inflammatory gene expression was determined by qPCR. Data are fromone experiment performed in triplicate and represent the means±SEM.

FIG. 17C is a set of bar graphs showing the results of HEK293T cellsthat were co-transfected ISRE or IFN-β promoter reporter plasmid (200ng) along with 5′pppRNA (10 ng/ml). Luciferase activity was analyzed 24h post-transfection by the Dual-Luciferase Reporter assay. Relativeluciferase activity was measured as fold induction relative to the basallevel of reporter gene. Data are from one experiment performed intriplicate and represent the means±SEM.

FIG. 17D is an image of an immunoblot showing the results of A549 cellsthat were transfected with 5′pppRNA (1 ng/ml) for 24 h. Whole cellextracts were prepared, resolved by SDS-PAGE, and analyzed byimmunoblotting for ISG56, STAT1, pIRF3 S396, IRF3, RIG-I, and β-actin.One representative Western blot from one experiment is shown.

FIG. 17E is a plot showing the results of A549 cells that weretransfected with 5′pppRNA (0.001-10 ng/ml) using Lipofectamine RNAiMaxfor 24 h then challenged with dengue virus (MOI 0.1) for 24 h.Percentage of infected cells was determined by intracellular staining ofDENV E protein expression. Data are from one experiment performed intriplicate and represent the means±SEM.

FIG. 18A is an image of an immunoblot showing the results of A549 cellsthat were transfected with control, RIG-I, TLR3, or MDA5 (30 pmol).After 48 h, M8 5′pppRNA (0.1 ng/ml) was transfected and 24 h aftertreatment, whole cell extracts were analyzed by SDS-PAGE andimmunoblotted for RIG-I, TLR3, MDA5, STAT1, ISG56, and β-actin. Onerepresentative Western blot out of three independent triplicates isshown.

FIG. 18B is a set of bar graphs showing the results of A549 cells thatwere transfected with siRNA and M8 5′pppRNA as in (18A), and antiviraland inflammatory gene expression was determined by qPCR. Data are fromone experiment performed in triplicate and represent the means±SEM.

FIG. 18C is an image of an immunoblot showing the results of A549 cellswere transfected with siRNA and M8 5′pppRNA as in (18A) then challengedwith H3N2 Brisbane A/59/2007 (MOI 0.2) for 24 h. Whole cell extractswere prepared, analyzed by SDS-PAGE, and immunoblotted for influenzaviral protein NS1 and β-actin. One representative Western blot out ofthree independent triplicates is shown.

FIG. 18D is a bar graph of viral titers in cell culture supernatants asdetermined by plaque assay. Data are from two independent experimentsperformed in triplicate and represent the means±SEM.

FIG. 18E is a bar graph showing the results of A549 cells that weretransfected with siRNA for the indicated pattern recognition receptorsand 5′pppRNA as in (18A) then challenged with dengue virus (MOI 0.1) for24 h. Percentage of infected cells was determined by intracellularstaining of DENV E protein expression. Data are from one experimentperformed in triplicate and represent the means±SEM.

FIG. 18F is a bar graph showing the results of DenV mRNA from cellsharvested from (FIG. 18E) that was quantified by qPCR. Data are from oneindependent experiment performed in triplicate and represent themeans±SEM.

FIG. 19A is a heatmap showing the results of Mo-DCs that weretransfected with 20 fmol of WT, M5, or M8 5′pppRNA or poly (I:C). After24 h, samples were analyzed by high throughput analysis of geneexpression by Fluidigm BioMark qPCR. Gene expression levels werecalculated using the ΔΔCt method and gene-wise standardized expression(z-score) was generated for each gene. The scale represents z-scorevalues where red shows an up-regulation and blue a down-regulation ingene expression. Heat map is representative of three individual donors.

FIG. 19B is a set of bar graphs showing selected genes from BioMark qPCRanalysis that are represented to show quantitative differences in RNAtreatment. Data are from three independent experiments and represent themeans±SEM.

FIG. 19C is a bar graph showing the results of Mo-DCs that weretransfected with WT, M5, or M8 5′pppRNA (10 ng/ml) for 24 h. pSTAT1expression is represented as geomean fluorescence as measured by flowcytometry analysis. Data are from one independent experiment performedin triplicate and represent the means±SEM.

FIG. 19D is an image of an immunoblot showing the results of Mo-DCs weretransfected with M8 5′pppRNA (10 ng/ml) for 24 h then challenged withinfluenza H3N2 Brisbane A/59/2007 (MOI 2) for 24 h. Whole cell extractswere prepared, analyzed by SDS-PAGE, and immunblotted for influenzaviral protein NS1 and β-actin. One representative Western blot from oneexperiment is shown.

FIG. 19E is a bar graph of viral titers in cell culture supernatantsthat were determined by plaque assay. Data are from one experimentperformed in triplicate and represent the means±SEM.

FIG. 19F is an image of an immunoblot showing the results of humanmonocyte-derived dendritic cells (Mo-DCs) that were transfected with WT,M5, or M8 5′pppRNA (10 ng/ml) using HiPerFect transfection reagent for24 h then challenged with dengue virus (MOI 10). Whole cell extractswere prepared, analyzed by SDS-PAGE, and immunoblotted for DenV viral Eprotein, STAT1, RIG-I, ISG56, and β-actin. One representative Westernblot from one experiment is shown.

FIG. 20A is an image of an immunoblot showing the results of A549 cellsthat were treated with oseltamivir phosphate (0.1 or 1 mM) ortransfected with M8 5′pppRNA (0.1 or 1 ng/ml) for 24 h then challengedwith the indicated strains of influenza (MOI 2). Whole cell extractswere prepared 24 h post-infection, subjected to SDS-PAGE, and probedwith antibodies for influenza viral protein NS1 and β-actin. Onerepresentative Western blot out of two independent experiments is shown.

FIG. 20B is a set of bar graphs of viral titers in cell culturesupernatants from (FIG. 20A) were determined by plaque assay. Data arefrom two independent experiments performed in triplicate and representthe means±SEM.

FIG. 21A is a bar graph of the results of A549 cells that weretransfected with M8 5′pppRNA (1 ng/ml) for 24 h then challenged withdengue virus (MOI 0.5). Percentage of infected cells was determined 24,48, and 72 h post-infection by intracellular staining of DENV E proteinexpression. Data are from one experiment performed in triplicate andrepresent the means±SEM.

FIG. 21B is an image of an immunoblot showing the results of A549 cellsthat were transfected with 5′pppRNA (0.1, 1 ng/ml) for 24 h thenchallenged with H3N2 Brisbane 59/2007 (MOI 0.2). Whole cell extractswere prepared at different times after transfection (24, 48, 72 h),subjected to SDS-PAGE, and probed with antibodies for influenza viralprotein NS1 and β-actin. One representative Western blot out of threeindependent triplicates is shown.

FIG. 21C is a bar graph showing viral titers in cell culturesupernatants that were determined by plaque assay. Data are from twoindependent experiments performed in triplicate and represent themeans±SEM.

FIG. 21D is a bar graph showing the results of A549 cells that wereinfected with dengue (MOI 0.1) for 1 h then transfected with M8 5′pppRNA(0.01 ng/ml) at 1, 4, and 8 h post infection. Percentage of infectedcells was determined 24 h post-infection by intracellular staining ofDENV E protein expression.

FIG. 21E is an image of an immunoblot showing the results of Mo-DCs thatwere infected with influenza (MOI 0.2) for 1 h followed by transfectionof M8 5′pppRNA at 1, 2, 4, and 8 h post-infection. Whole cell extractswere prepared 24 h post-infection, subjected to SDS-PAGE, and probedwith antibodies for NS1 and β-actin. One representative Western blot outof three independent triplicates is shown.

FIG. 22A is a plot showing the survival of BALB/c mice (n=5) that wereinjected IV with 5 μg of WT, M5 or M8 5′pppRNA complexed with invivo-JetPEI one day prior to and on the day of infection with H5N1-REinfluenza (5,000 PFU).

FIG. 22B is a plot showing the weight loss of BALB/c mice treated as inFIG. 22A. FIG. 22C is a plot showing the sickness score of BALB/c micetreated as in FIG. 22A.

FIG. 22D is a bar graph showing the results of H5N1-RE viral replicationin lungs of animals treated with WT, M5 or M8 was quantified by plaqueassay at days 1, 3 and 5. Error bars indicate SEM for five animals.

FIG. 22E is a plot showing the results where control RNA or M8 5′pppRNA(2 μg) complexed with in vivo JetPEI was injected intramuscularly intoadult mice on the day prior to and day of viral infection. Mice wereinfected with chikungunya via footpad injection. Footpad swelling wasmonitored and measured daily by caliper during the course of 14 days.

FIG. 22F is a bar graph of serum viral load at day 3 for the animalstreated as described in FIG. 22E.

Data in FIGS. 23A and 23B were obtained by transfecting monocyte-deriveddendritic cells (MDDCs) with WT, M5, or M8 5′pppRNA or poly(I:C)increases expression of activation and differentiation markers and theirmRNA levels. MDDC were isolated from peripheral blood mononuclear cells(n=4), differentiated, and transfected with 10 ng WT, M5, M8, orpoly(I:C) using HiPerFect transfection reagent for 24 h.

FIG. 23A is a heat map showing the results of gene expression analysisusing the Fluidigm BioMark platform for the indicated genes in MDDCstransfected with 20 fmol of WT, M5, M8, or poly(I:C) for 24 h.

FIG. 23B is a set of four bar graphs showing the surface expression ofthe indicated activation and differentiation markers as assessed by flowcytometry (mean±SEM); *P≤0.05; **P≤0.01; ***P≤0.005.

FIGS. 24A-24D summarize the Protective efficacy of a VLP vaccineadjuvanted with M5, M8 or poly(I:C). Mice (n=5) were immunizedintramuscularly with 2 μg of VLP alone or combined with 5 μg M5, M8 orpoly(I:C) as a 50 μL injection. Three weeks later the mice werechallenged with 5,000 pfu of H5N1.

FIG. 24A is a bar graph showing hemagglutination inhibition (HAI)antibody titers in immunized mice prior to infection were determined byhemagglutination inhibition assay using horse red blood cells.

FIG. 24B is a bar graph showing the results of an assessment of viralreplication in lungs of infected animals 3 days post infection by plaqueassay.

FIG. 24C is a bar graph showing TUNEL-positive (apoptotic) lung cells ininfected mice were quantified using a TUNEL assay.

FIG. 24D is a set of eight images showing H&E staining of paraffinembedded lung cross-sections from mice 3 days after challenge. Yellowarrow indicates airways of mice that were vaccinated with either M8-only(top) or M8-VLP (bottom). All values are expressed as the mean±SEM.*P≤0.05; ***P≤0.005.

FIG. 25A is a bar graph showing the dose-response to VLP and M8 by HAIantibody titer. Mice (n=5) were immunized with the indicated doses ofVLP (2 μg-0.5 μg) in combination with 5 μg of M8. Three weeks later,mice were challenged with H5N1, and lungs from infected animals wereharvested 3 days post-challenge. HAI antibody titers in immunized miceprior to infection were determined by HAI assay.

FIG. 25B is a bar graph showing the dose-response to VLP and M8 by viralplaque assay. Mice (n=5) were immunized with decreasing doses of VLP (2μg-0.5 μg) in combination with 5 μg of M8, three weeks later challengedwith H5N1, and lungs from infected animals were harvested 3 dayspost-challenge. Viral replication in lungs was assessed by plaque assay.

For FIGS. 25C-25F, Mice were immunized with 0.5 μg of VLP with 0.1-5 μgof M8. HAI antibody titers were determined by HAI assay.

FIG. 25C is a bar graph of influenza HAI antibody titers.

FIG. 25D is a bar graph of viral replication in lungs by plaque assay.FIG. 25E is a plot of weight loss in the mice over time.

FIG. 25F is a plot of survival of the mice over time.

FIG. 26A-26D collectively show an adjuvant comparison strategy andantibody immune responses for M8, Alum, AddaVax, andpoly(I:C)-adjuvanted VLP vaccine.

FIG. 26A is a figure describing the strategy for adjuvant comparison.

For FIGS. 26B-26D, Mice were immunized with 0.5 μg of VLP in combinationwith 5 μg of M8 or poly(I:C), or in combination with 50% volume of Alumor AddaVax, and five days and three weeks after immunization sera werecollected.

FIG. 26B is a bar graph showing HA-specific IgG antibodies determined 3weeks after immunization by ELISA.

FIG. 26C is a bar graph showing Influenza HAI antibody titers determined3 weeks after immunization by HAI assay.

FIG. 26D is a plot of HA-specific IgM antibodies were determined fivedays after immunization by ELISA. All values are expressed as themean±SEM. *P≤0.05; **P≤0.01; ***P≤0.005; O.D., optical density; 3 d,three days.

FIG. 27A-27D collectively show the protective efficacy of 0.5 μg of VLPin combination with 5 g of M8 or poly(I:C), or in combination with 50%volume of Alum or AddaVax. Three weeks after vaccination mice (n=8) werechallenged with the lethal dose of H5N1 (5,000 pfu). All values areexpressed as the mean±SEM. ***P≤0.005.

FIG. 27A is a plot showing survival. FIG. 27B is a plot showing weight.

FIG. 27C is a plot showing sickness score.

FIG. 27D is a bar graph showing viral replication in lungs was assessedby plaque assay in a separate group of immunized animals than thatdescribed above (n=5) 3 days post-infection.

FIGS. 28A-28E collectively show the long-term protective responses inmice immunized with 0.5 μg of VLP in combination with 5 μg of M8 orpoly(I:C), or in combination with 50% volume of Alum or AddaVax. Mousesera (n=8) were collected 4 (white bars) and 16 weeks (black bars)post-vaccination to determine HA-specific IgG antibodies (ELISA).

FIG. 28A is a bar graph showing HA-specific IgG antibodies by ELISA.FIG. 28B is a bar graph showing HAI antibody titer by HAI assay.

FIG. 28C is a plot showing weight of animals after challenge with alethal H5N1 dose (5000 pfu).

FIG. 28D is a plot showing survival of animals after challenge with alethal H5N1 dose (5000 pfu).

FIG. 28E is a plot showing sickness score of animals after challengewith a lethal H5N1 dose (5000 pfu.)

FIG. 29A-29C collectively show the quantification of germinal center(GC) B cells from spleens of IM immunized mice (n=5) by flow cytometry.

FIG. 29A illustrates the gating strategy for quantification of GC Bcells. FIG. 29B is a bar graph showing the percent GC B cells inB220^(hi) splenocytes.

FIG. 29C is a bar graph showing the quantification of IgG1⁺ GC B cells.All values are expressed as the mean±SEM. *P≤0.05; **P≤0.01; ***P≤0.005.

FIG. 30A-30F collectively show the quantification of IgG subclasses fromIM vaccinated animals (n=8) and intracellular cytokine levels in T cellsisolated from spleens of IP vaccinated animals (n=5) after 24 h of VLPstimulation. All values are expressed as the mean±SEM. *P≤0.05;**P≤0.01; ***P≤0.005

FIG. 30A is a bar graph of IgG subclasses, IgG1, IgG2a, IgG2b, and IgG3,from sera of vaccinated animals determined by ELISA using HA-coatedplates.

FIG. 30B is a bar graph of the percent of IFNγ⁺ CD8^(hi) cells.

FIG. 30C is a bar graph of the percent of IL-2⁺ CD4^(hi) cells.

FIG. 30D is a bar graph of the percent of TNFα⁺ CD4^(hi) cells.

FIG. 30E is a bar graph of the percent of IFNγ⁺ CD4^(hi) cells.

FIG. 30F is a bar graph of the percent of IL-10⁺ CD4^(hi) cells.

SEQUENCE LISTING

SEQ ID NO: 1 is a polynucleotide that makes up part of anoligoribonucleotide described herein.

SEQ ID NO: 2 is a polynucleotide that makes up part of anoligoribonucleotide described herein.

SEQ ID NO: 3 is a polynucleotide that makes up part of anoligoribonucleotide described herein.

SEQ ID NO: 4 is a polynucleotide that makes up part of anoligoribonucleotide described herein.

SEQ ID NO: 5 is an oligoribonucleotide. 5′pppSEQ ID NO: 5 is alsoreferred to as WT (wild type) herein.

SEQ ID NO: 6 is an oligoribonucleotide. 5′pppSEQ ID NO: 6 is alsoreferred to as M1 herein.

SEQ ID NO: 7 is an oligoribonucleotide. 5′pppSEQ ID NO: 7 is alsoreferred to as M2 herein.

SEQ ID NO: 8 is an oligoribonucleotide. 5′pppSEQ ID NO: 8 is alsoreferred to as M3 herein.

SEQ ID NO: 9 is an oligoribonucleotide. 5′pppSEQ ID NO: 9 is alsoreferred to as M4 herein.

SEQ ID NO: 10 is an oligoribonucleotide. 5′pppSEQ ID NO: 10 is alsoreferred to as M5 herein.

SEQ ID NO: 11 is an oligoribonucleotide. 5′pppSEQ ID NO: 11 is alsoreferred to as M6 herein.

SEQ ID NO: 12 is an oligoribonucleotide. 5′pppSEQ ID NO: 12 is alsoreferred to as M7 herein.

SEQ ID NO: 13 is an oligoribonucleotide. 5′pppSEQ ID NO: 13 is alsoreferred to as M8 herein.

SEQ ID NO: 14 is an oligoribonucleotide. 5′pppSEQ ID NO: 14 is alsoreferred to as M8A herein.

SEQ ID NO: 15 is an oligoribonucleotide. 5′pppSEQ ID NO: 15 is alsoreferred to as M8B herein.

SEQ ID NO: 16 is an oligoribonucleotide. 5′pppSEQ ID NO: 16 is alsoreferred to as M8C herein.

SEQ ID NO: 17 is an oligoribonucleotide. 5′pppSEQ ID NO: 17 is alsoreferred to as M8D herein.

SEQ ID NO: 18 is a VSV WT forward primer.

SEQ ID NO: 19 is a VSV WT reverse primer.

SEQ ID NO: 20 is an M1 forward primer.

SEQ ID NO: 21 is an M1 reverse primer.

SEQ ID NO: 22 is an M2 forward primer.

SEQ ID NO: 23 is an M2 reverse primer.

SEQ ID NO: 24 is an M3 forward primer.

SEQ ID NO: 25 is an M3 reverse primer.

SEQ ID NO: 26 is an M4 forward primer.

SEQ ID NO: 27 is an M4 reverse primer.

SEQ ID NO: 28 is an M5 forward primer.

SEQ ID NO: 29 is an M5 reverse primer.

SEQ ID NO: 30 is an M6 forward primer.

SEQ ID NO: 31 is an M6 reverse primer.

SEQ ID NO: 32 is an M7 forward primer.

SEQ ID NO: 33 is an M7 reverse primer.

SEQ ID NO: 34 is an M8 forward primer.

SEQ ID NO: 35 is an M8 reverse primer.

SEQ ID NO: 36 is an M8A forward primer.

SEQ ID NO: 37 is an M8A reverse primer.

SEQ ID NO: 38 is an M8B forward primer.

SEQ ID NO: 39 is an M8B reverse primer.

SEQ ID NO: 40 is an M8C forward primer.

SEQ ID NO: 41 is an M8C reverse primer.

SEQ ID NO: 42 is an M8D forward primer.

SEQ ID NO: 43 is an M8D reverse primer.

SEQ ID NO: 44 is a BIRC3 forward primer.

SEQ ID NO: 45 is a BIRC3 reverse primer.

SEQ ID NO: 46 is a CCL3 forward primer.

SEQ ID NO: 47 is a CCL3 reverse primer.

SEQ ID NO: 48 is a CCL5 forward primer.

SEQ ID NO: 49 is a CCL5 reverse primer.

SEQ ID NO: 50 is a CXCL10 forward primer.

SEQ ID NO: 51 is a CXCL10 reverse primer.

SEQ ID NO: 52 is a DDX58 forward primer.

SEQ ID NO: 53 is a DDX58 reverse primer.

SEQ ID NO: 54 is a DENV2 forward primer.

SEQ ID NO: 55 is a DENV2 reverse primer.

SEQ ID NO: 56 is a GADPH forward primer.

SEQ ID NO: 57 is a GADPH reverse primer.

SEQ ID NO: 58 is an IFIT1 forward primer.

SEQ ID NO: 59 is an IFIT1 reverse primer.

SEQ ID NO: 60 is an IFIT2 forward primer.

SEQ ID NO: 61 is an IFIT2 reverse primer.

SEQ ID NO: 62 is an IFITM1 forward primer.

SEQ ID NO: 63 is an IFITM1 reverse primer.

SEQ ID NO: 64 is an IFITM2 forward primer.

SEQ ID NO: 65 is an IFITM2 reverse primer.

SEQ ID NO: 66 is an IFITM3 forward primer.

SEQ ID NO: 67 is an IFITM3 reverse primer.

SEQ ID NO: 68 is an IFNAR1 forward primer.

SEQ ID NO: 69 is an IFNAR1 reverse primer.

SEQ ID NO: 70 is an IFNAR2 forward primer.

SEQ ID NO: 71 is an IFNAR2 reverse primer.

SEQ ID NO: 72 is an IFNB1 forward primer.

SEQ ID NO: 73 is an IFNB1 reverse primer.

SEQ ID NO: 74 is an IL1A forward primer.

SEQ ID NO: 75 is an IL1A reverse primer.

SEQ ID NO: 76 is an IL1B forward primer.

SEQ ID NO: 77 is an IL1B reverse primer.

SEQ ID NO: 78 is an IL-6 forward primer.

SEQ ID NO: 79 is an IL-6 reverse primer.

SEQ ID NO: 80 is an IL-8 forward primer.

SEQ ID NO: 81 is an IL-8 reverse primer.

SEQ ID NO: 82 is an IL-10 forward primer.

SEQ ID NO: 83 is an IL-10 reverse primer.

SEQ ID NO: 84 is an IL-12A forward primer.

SEQ ID NO: 85 is an IL-12A reverse primer.

SEQ ID NO: 86 is an IL-28RA forward primer.

SEQ ID NO: 87 is an IL-28RA reverse primer.

SEQ ID NO: 88 is an IL-29 forward primer.

SEQ ID NO: 89 is an IL-29 reverse primer.

SEQ ID NO: 90 is an IRF3 forward primer.

SEQ ID NO: 91 is an IRF3 reverse primer.

SEQ ID NO: 92 is an IRF7 forward primer.

SEQ ID NO: 93 is an IRF7 reverse primer.

SEQ ID NO: 94 is an ISG15 forward primer.

SEQ ID NO: 95 is an ISG15 reverse primer.

SEQ ID NO: 96 is an MX1 forward primer.

SEQ ID NO: 97 is an MX1 reverse primer.

SEQ ID NO: 98 is an MX2 forward primer.

SEQ ID NO: 99 is an MX2 reverse primer.

SEQ ID NO: 100 is an SOCS3 forward primer.

SEQ ID NO: 101 is an SOCS3 reverse primer.

SEQ ID NO: 102 is a STAT1 forward primer.

SEQ ID NO: 103 is a STAT1 reverse primer.

SEQ ID NO: 104 is a TANK forward primer.

SEQ ID NO: 105 is a TANK reverse primer.

SEQ ID NO: 106 is a TLR3 forward primer.

SEQ ID NO: 107 is a TLR3 reverse primer.

SEQ ID NO: 108 is a TLR7 forward primer.

SEQ ID NO: 109 is a TLR7 reverse primer.

SEQ ID NO: 110 is a TNF forward primer.

SEQ ID NO: 111 is a TNF reverse primer.

SEQ ID NO: 112 is a CD40 forward primer.

SEQ ID NO: 113 is a CD40 reverse primer.

SEQ ID NO: 114 is a CD74 forward primer.

SEQ ID NO: 115 is a CD74 reverse primer.

SEQ ID NO: 116 is a CD80 forward primer.

SEQ ID NO: 117 is a CD80 reverse primer.

SEQ ID NO: 118 is a CD83 forward primer.

SEQ ID NO: 119 is a CD83 reverse primer.

SEQ ID NO: 120 is a CD86 forward primer.

SEQ ID NO: 121 is a CD86 reverse primer.

SEQ ID NO: 122 is a 4-1BB forward primer.

SEQ ID NO: 123 is a 4-1BB reverse primer.

SEQ ID NO: 124 is an HLA-DRA forward primer.

SEQ ID NO: 125 is an HLA-DRA reverse primer.

SEQ ID NO: 126 is an HLA-DQA forward primer.

SEQ ID NO: 127 is an HLA-DQA reverse primer.

DETAILED DESCRIPTION

Disclosed herein are synthetic oligoribonucleotides, each synthesizedwith a triphosphate group at its 5′ end. The oligoribonucleotideincludes: a first polynucleotide of SEQ ID NO: 1, a secondpolynucleotide of SEQ ID NO: 2 and a third polynucleotide of SEQ ID NO:3 with SEQ ID NO: 3 located between SEQ ID NO: 1 and SEQ ID NO: 2. SEQID NO: 1 can be 5′ of SEQ ID NO: 2 or SEQ ID NO: 1 can be 3′ of SEQ IDNO: 2. The oligoribonucleotides can comprise any additional sequence.

In examples where SEQ ID NO: 1 is 5′ of SEQ ID NO: 2, theoligoribonucleotides comprise the structure:GACGAAGACCACAAAACCAGAU(A)_(n)UAA(U)_(n)AUCUGGUUUUGUGGUCUUCGUC orGACGAAGACCACAAAACCAGAU(U)_(n)UAA(A)_(n)AUCUGGUUUUGUGGUCUUCGUC; wherein nis any integer greater than 1. This structure indicates that thenucleotide in parentheses is repeated the number of times equal to n.For example, n can equal 2, 3, 6, 11, 16, 26, or more that 26 repeats ofthe nucleotide indicated in parentheses. In this example, the number ofA or U nucleotides is equal.

The oligoribonucleotides can also comprise the structure:GACGAAGACCACAAAACCAGAU(AAU)_(x)U(AUU)_(y)AUCUGGUUUUGUGGUCUUCGUC orGACGAAGACCACAAAACCAGAU(AUU)_(x)U(AAU)_(y)AUCUGGUUUUGUGGUCUUCGUC; whereinx and y are any integer greater than 2. In this example the tripeptidein parentheses is repeated a number of times equal to x or y. In thisexample, x and y can be different numbers. For example x can equal 10while y can equal 8.

In examples where SEQ ID NO: 1 is 3′ of SEQ ID NO: 2, theoligoribonucleotides can have the structure:AUCUGGUUUUGUGGUCUUCGUC(A)_(n)UAA(U)_(n)GACGAAGACCACAAAACCAGAU; orAUCUGGUUUUGUGGUCUUCGUC(U)_(n)UAA(A)_(n)GACGAAGACCACAAAACCAGAU, wherein nis an integer greater than 1.

Alternatively, the synthetic oligoribonucleotide can be anoligoribonucleotide of at least 59 nucleotides in length that can form ahairpin structure comprising at least 29 base pairs, the syntheticoligonucleotide further comprising a triphosphate group at the 5′ end ofthe oligoribonucleotide. In examples of these aspects, theoligoribonucleotides are at least 99 nucleotides in length that can forma hairpin structure comprising at least 49 base pairs.

The synthetic oligoribonucleotides described herein can be expressedfrom a DNA plasmid. Such a DNA plasmid comprises the DNA sequence thatencodes the described oligoribonucleotides. The oligoribonucleotides canbe transcribed as an RNA molecule that automatically folds into duplexeswith hairpin loops. Typically, a transcriptional unit or cassette willcontain an RNA transcript promoter sequence, such as a T7 promoteroperably linked to the sequence encoding the oligoribonucleotide.

The synthetic oligoribonucleotides described herein comprise a5′-triphosphate group. These may collectively be referred to as 5′pppRNAor individually as 5′pppSEQ ID NO: XX herein. Alternatively, individualcompounds may be referred to herein by names such as WT, M5, or M8 asindicated in the Sequence Listing above.

Methods of isolating RNA, synthesizing RNA, hybridizing nucleic acids,making and screening cDNA libraries, and performing PCR are well knownin the art (see, e.g., Gubler and Hoffman, Gene 25, 263-269 (1983);Sambrook and Russell, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor N.Y., (2001)) as arePCR methods (see, U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols:A Guide to Methods and Applications, Innis et al, Eds, (1990)).Expression libraries are also well known to those of skill in the art.Additional basic texts disclosing the general methods of use in thisinvention include Sambrook and Russell (2001) supra; Kriegler, GeneTransfer and Expression: A Laboratory Manual (1990); and CurrentProtocols in Molecular Biology (Ausubel et al., eds., 1994).

An oligoribonucleotide can also be chemically synthesized. Synthesis ofthe single-stranded nucleic acid makes use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-endand phosphoramidites at the 3′-end. As a non-limiting example, smallscale syntheses can be conducted on an Applied Biosystems synthesizerusing a 0.2 micromolar scale protocol with a 2.5 min coupling step for2′-O-methylated nucleotides. Alternatively, syntheses at the 0.2micromolar scale can be performed on a 96-well plate synthesizer fromProtogene. However, a larger or smaller scale of synthesis isencompassed by the invention, including any method of synthesis nowknown or yet to be disclosed. Suitable reagents for synthesis of thesingle-stranded oligonucleotides, methods of RNA deprotection, methodsof RNA purification, and methods of adding phosphate groups to anoligoribonucleotide are known to those of skill in the art.

An oligoribonucleotide can be synthesized via a tandem synthesistechnique, wherein both strands are synthesized as a single continuousfragment or strand separated by a linker that is subsequently cleaved toprovide separate fragments or strands that hybridize to form an RNAduplex. The linker can be any linker, including a polynucleotide linkeror a non-nucleotide linker. The linker can comprise any sequence of oneor more ribonucleotides. The tandem synthesis of RNA can be readilyadapted to both multiwell/multiplate synthesis platforms as well aslarge scale synthesis platforms employing batch reactors, synthesiscolumns, and the like.

Alternatively, the oligoribonucleotide can be assembled from twodistinct single-stranded molecules, wherein one strand includes thesense strand and the other includes the antisense strand of the RNA. Forexample, each strand can be synthesized separately and joined togetherby hybridization or ligation following synthesis and/or deprotection.Either the sense or the antisense strand can contain additionalnucleotides that are not complementary to one another and do not form adouble stranded RNA molecule. In certain other instances, theoligoribonucleotide can be synthesized as a single continuous fragment,where the self-complementary sense and antisense regions hybridize toform an RNA duplex having a hairpin or panhandle secondary structure.

An oligoribonucleotide can comprise a duplex having two complementarystrands that form a double-stranded region with least one modifiednucleotide in the double-stranded region. The modified nucleotide may beon one strand or both. If the modified nucleotide is present on bothstrands, it may be in the same or different positions on each strand.Examples of modified nucleotides suitable for use in the presentinvention include, but are not limited to, ribonucleotides having a2′-O-methyl (2′OMe), 2′-deoxy-2′-fluoro (2′F), 2′-deoxy, 5-C-methyl,2′-O-(2-methoxyethyl) (MOE), 4′-thio, 2′-amino, or 2′-C-allyl group.Modified nucleotides having a conformation such as those described in,for example in Sanger, Principles of Nucleic Acid Structure,Springer-Verlag Ed. (1984), are also suitable for use inoligoribonucleotides. Other modified nucleotides include, withoutlimitation: locked nucleic acid (LNA) nucleotides, G-clamp nucleotides,or nucleotide base analogs. LNA nucleotides include but need not belimited to 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides),2′-O-(2-methoxyethyl) (MOE) nucleotides, 2′-methyl-thio-ethylnucleotides, 2′-deoxy-2′-fluoro (2′F) nucleotides, 2′-deoxy-2′-chloro(2Cl) nucleotides, and 2′-azido nucleotides. A G-clamp nucleotide refersto a modified cytosine analog wherein the modifications confer theability to hydrogen bond both Watson-Crick and Hoogsteen faces of acomplementary guanine nucleotide within a duplex (Lin et al, J Am ChemSoc, 120, 8531-8532 (1998)). Nucleotide base analogs include forexample, C-phenyl, C-naphthyl, other aromatic derivatives, inosine,azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole,4-nitroindole, 5-nitroindole, and 6-nitroindole (Loakes, Nucl Acids Res,29, 2437-2447 (2001)).

An oligoribonucleoitde can comprise one or more chemical modificationssuch as terminal cap moieties, phosphate backbone modifications, and thelike. Examples of classes of terminal cap moieties include, withoutlimitation, inverted deoxy abasic residues, glyceryl modifications,4′,5′-methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides,4′-thio nucleotides, carbocyclic nucleotides, 1,5-anhydrohexitolnucleotides, L-nucleotides, α-nucleotides, modified base nucleotides,threo pentofuranosyl nucleotides, acyclic 3′,4′-seco nucleotides,acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentylnucleotides, 3′-3′-inverted nucleotide moieties, 3′-3′-inverted abasicmoieties, 3′-2′-inverted nucleotide moieties, 3′-2′-inverted abasicmoieties, 5′-5′-inverted nucleotide moieties, 5′-5′-inverted abasicmoieties, 3′-5′-inverted deoxy abasic moieties, 5′-amino-alkylphosphate, 1,3-diamino-2-propyl phosphate, 3 aminopropyl phosphate,6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropylphosphate, 1,4-butanediol phosphate, 3′-phosphoramidate, 5′phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate,5′-amino, 3′-phosphorothioate, 5′-phosphorothioate, phosphorodithioate,and bridging or non-bridging methylphosphonate or 5′-mercapto moieties(see, e.g., U.S. Pat. No. 5,998,203; Beaucage et al, Tetrahedron 49,1925 (1993)). Non-limiting examples of phosphate backbone modifications(i.e., resulting in modified internucleotide linkages) includephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate, carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker et al,Modern Synthetic Methods, VCH, 331-417 (1995); Mesmaeker et al,Antisense Research, ACS, 24-39 (1994)). Such chemical modifications canoccur at the 5′-end and/or 3′-end of the sense strand, antisense strand,or both strands of the oligoribonucleotide.

The sense and/or antisense strand of an oligoribonucleotide may comprisea 3′-terminal overhang having 1 to 4 or more 2′-deoxyribonucleotidesand/or any combination of modified and unmodified nucleotides.Additional examples of modified nucleotides and types of chemicalmodifications that can be introduced into the modifiedoligoribonucleotides of the present invention are described, e.g., in UKPatent No. GB 2,397,818 B and U.S. Patent Publication Nos. 20040192626and 20050282188.

An oligoribonucleotide may comprise one or more non-nucleotides in oneor both strands of the siRNA. A non-nucleotide can be any subunit,functional group, or other molecular entity capable of beingincorporated into a nucleic acid chain in the place of one or morenucleotide units that is not or does not comprise a commonly recognizednucleotide base such as adenosine, guanine, cytosine, uracil, orthymine, such as a sugar or phosphate.

Chemical modification of the disclosed oligoribonucleotides may alsocomprise attaching a conjugate to the oligoribonucleotide molecule. Theconjugate can be attached at the 5′- and/or the 3′-end of the senseand/or the antisense strand of the oligoribonucleotide via a covalentattachment such as a nucleic acid or non-nucleic acid linker. Theconjugate can also be attached to the oligoribonucleotide through acarbamate group or other linking group (see, e.g., U.S. PatentPublication Nos. 20050074771, 20050043219, and 20050158727). A conjugatemay be added to the oligoribonucleotide for any of a number of purposes.For example, the conjugate may be a molecular entity that facilitatesthe delivery of the oligoribonucleotide into a cell or the conjugate amolecule that comprises a drug or label.

Examples of conjugate molecules suitable for attachment to the disclosedoligoribonucleotides include, without limitation, steroids such ascholesterol, glycols such as polyethylene glycol (PEG), human serumalbumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates(e.g., folic acid, folate analogs and derivatives thereof), sugars(e.g., galactose, galactosamine, N-acetyl galactosamine, glucose,mannose, fructose, fucose, etc.), phospholipids, peptides, ligands forcellular receptors capable of mediating cellular uptake, andcombinations thereof (see, e.g., U.S. Patent Publication Nos.20030130186, 20040110296, and 20040249178; U.S. Pat. No. 6,753,423).Other examples include the lipophilic moiety, vitamin, polymer, peptide,protein, nucleic acid, small molecule, oligosaccharide, carbohydratecluster, intercalator, minor groove binder, cleaving agent, andcross-linking agent conjugate molecules described in U.S. PatentPublication Nos. 20050119470 and 20050107325. Other examples include the2′-O-alkyl amine, 2′-O-alkoxyalkyl amine, polyamine, C5-cationicmodified pyrimidine, cationic peptide, guanidinium group, amidininiumgroup, cationic amino acid conjugate molecules described in U.S. PatentPublication No. 20050153337. Additional examples of conjugate moleculesinclude a hydrophobic group, a membrane active compound, a cellpenetrating compound, a cell targeting signal, an interaction modifier,or a steric stabilizer as described in U.S. Patent Publication No.20040167090. Further examples include the conjugate molecules describedin U.S. Patent Publication No. 20050239739.

The type of conjugate used and the extent of conjugation to thedisclosed oligoribonucleotides can be evaluated for improvedpharmacokinetic profiles, bioavailability, and/or stability of theoligoribonucleotide while retaining activity. As such, one skilled inthe art can screen oligoribonucleotides having various conjugatesattached thereto to identify oligonucleotide conjugates having improvedproperties using any of a variety of well-known in vitro cell culture orin vivo animal models.

Pharmaceutical Compositions

An oligoribonucleotide may be incorporated into a pharmaceuticallyacceptable carrier or transfection reagent containing theoligoribonucleotides described herein. The carrier system may be alipid-based carrier system such as a stabilized nucleic acid-lipidparticle (e.g., SNALP or SPLP), cationic lipid or liposome nucleic acidcomplexes (i.e., lipoplexes), a liposome, a micelle, a virosome, or amixture thereof. In other embodiments, the carrier system is apolymer-based carrier system such as a cationic polymer-nucleic acidcomplex (i.e., polyplex). In additional embodiments, the carrier systemis a cyclodextrin-based carrier system such as a cyclodextrinpolymer-nucleic acid complex (see US Patent Application Publication20070218122). In further embodiments, the carrier system is aprotein-based carrier system such as a cationic peptide-nucleic acidcomplex. An oligoribonucleotide molecule may also be delivered as nakedRNA.

A pharmaceutical composition can be any combination of active and/orinert materials that can be administered to a subject for the purpose oftreating a disease. A pharmaceutically acceptable carrier(interchangeably termed a vehicle) can be any material or molecularentity that facilitates the administration or other delivery of thepharmaceutical composition. In general, the nature of the carrier willdepend on the particular mode of administration being employed. Forinstance, parenteral formulations usually comprise injectable fluidsthat include pharmaceutically and physiologically acceptable fluids suchas water, physiological saline, balanced salt solutions, aqueousdextrose, glycerol or the like as a vehicle.

A pharmaceutical composition can comprise an adjuvant. An adjuvant canbe any compound, composition, or substance that when used in combinationwith an immunogenic agent augments or otherwise alters or modifies aresultant immune response. In some examples, an adjuvant increases thetiter of antibodies induced in a subject by the immunogenic agent. Inanother example, if the antigenic agent is a multivalent antigenicagent, an adjuvant alters the particular epitopic sequences that arespecifically bound by antibodies induced in a subject. In some examples,the oligoribonucleotides are added to an immunogenic agent (such as H5N1virus-like particles) to act as an adjuvant.

Other agents that can be used as adjuvants in formulating pharmaceuticalcompositions used to produce an immune response include Freund'sIncomplete Adjuvant (IFA), Freund's complete adjuvant, B30-MDP,LA-15-PH, montanide, saponin, aluminum salts such as aluminum hydroxide(Amphogel, Wyeth Laboratories, Madison, N.J.), alum, lipids, the MF59microemulsion, a mycobacterial antigen, vitamin E, non-ionic blockpolymers, muramyl dipeptides, polyanions, amphipatic substances, ISCOMs(immune stimulating complexes, such as those disclosed in EuropeanPatent EP 109942), vegetable oil, Carbopol, aluminium oxide,oil-emulsions (such as Bayol F or Marcol 52), E. coli heat-labile toxin(LT), Cholera toxin (CT), and combinations thereof. Such adjuvants canbe formulated with the disclosed oligoribonucleotides.

A pharmaceutical composition comprising an immunogenic protein and anadjuvant can also be termed a vaccine. In some examples, the immunogenicprotein is a virus like particle. Virus-like particles (VLPs) arevaccine platforms composed of viral proteins that spontaneouslyassemble, mimicking the live virus but lacking genetic material andtherefore without the ability to replicate. Merck's human papillomavirus (HPV) vaccine Gardasil® is a quadrivalent HPV VLP vaccine thatprevents infection by HPV types 6, 11, 16, and 18, and contains Alum asadjuvant.

A therapeutically effective amount or concentration of a compound suchas the disclosed oligoribonucleotides may be any amount of a compositionthat alone, or together with one or more additional therapeutic agents,is sufficient to achieve a desired effect in a subject. The effectiveamount of the agent will be dependent on several factors, including, butnot limited to, the subject being treated and the manner ofadministration of the therapeutic composition. In one example, atherapeutically effective amount or concentration is one that issufficient to prevent advancement, delay progression, or to causeregression of a disease, or which is capable of reducing symptoms causedby any disease, including viral infection.

In one example, a desired effect is to reduce or inhibit one or moresymptoms associated with viral infection. The one or more symptoms donot have to be completely eliminated for the composition to beeffective. For example, a composition can decrease the sign or symptomby a desired amount, for example by at least 20%, at least 50%, at least80%, at least 90%, at least 95%, at least 98%, or even at least 100%, ascompared to the sign or symptom in the absence of the composition.

A therapeutically effective amount of a pharmaceutical composition canbe administered in a single dose, or in several doses, for exampledaily, during a course of treatment. However, the therapeuticallyeffective amount can depend on the subject being treated, the severityand type of the condition being treated, and the manner ofadministration. For example, a therapeutically effective amount of suchagent can vary from about 100 μg-10 mg per kg body weight ifadministered intravenously.

The actual dosages will vary according to factors such as the type ofvirus to be protected against and the particular status of the subject(for example, the subject's age, size, fitness, extent of symptoms,susceptibility factors, and the like) time and route of administration,other drugs or treatments being administered concurrently, as well asthe specific pharmacology of treatments for viral infection foreliciting the desired activity or biological response in the subject.Dosage regimens can be adjusted to provide an optimum prophylactic ortherapeutic response.

A therapeutically effective amount is also one in which any toxic ordetrimental side effects of the compound and/or other biologicallyactive agent is outweighed in clinical terms by therapeuticallybeneficial effects. A non-limiting range for a therapeutically effectiveamount of treatments for viral infection within the methods andformulations of the disclosure is about 0.0001 μg/kg body weight toabout 10 mg/kg body weight per dose, such as about μg/kg body weight toabout 0.001 μg/kg body weight per dose, about 0.001 μg/kg body weight toabout 0.01 μg/kg body weight per dose, about 0.01 μg/kg body weight toabout 0.1 μg/kg body weight per dose, about 0.1 μg/kg body weight toabout 10 μg/kg body weight per dose, about 1 μg/kg body weight to about100 μg/kg body weight per dose, about 100 μg/kg body weight to about 500μg/kg body weight per dose, about 500 μg/kg body weight per dose toabout 1000 μg/kg body weight per dose, or about 1.0 mg/kg body weight toabout 10 mg/kg body weight per dose.

Dosage can be varied by the attending clinician to maintain a desiredconcentration. Higher or lower concentrations can be selected based onthe mode of delivery, for example, trans-epidermal, rectal, oral,pulmonary, intranasal delivery, intravenous or subcutaneous delivery.

Determination of effective amount is typically based on animal modelstudies followed up by human clinical trials and is guided byadministration protocols that significantly reduce the occurrence orseverity of targeted disease symptoms or conditions in the subject.Suitable models in this regard include, for example, murine, rat,porcine, feline, non-human primate, and other accepted animal modelsubjects known in the art. Alternatively, effective dosages can bedetermined using in vitro models (for example, viral titer assays orcell culture infection assays). Using such models, only ordinarycalculations and adjustments are required to determine an appropriateconcentration and dose to administer a therapeutically effective amountof the treatments for viral infection (for example, amounts that areeffective to alleviate one or more symptoms of viral infection).

Methods of Treating Viral Infections

Disclosed herein are methods of treating a subject that has or may havea viral infection comprising administering a pharmaceutical compositioncomprising the disclosed oligoribonucleotides to the subject. Thesubject may be treated therapeutically or prophylactically.

A subject can be any multi-cellular vertebrate organisms, a categorythat includes human and non-human mammals, such as mice. In someexamples a subject is a male. In some examples a subject is a female.Further types of subjects to which the pharmaceutical composition may beproperly administered include subjects known to have a viral infection(through, for example, a molecular diagnostic test or clinicaldiagnosis,) subjects having a predisposition to contracting a viralinfection (for example by living in or travelling to a region in whichone or more viruses is endemic), or subjects displaying one or moresymptoms of having a viral infection.

Administration of a pharmaceutical composition may be any method ofproviding or give a subject a pharmaceutical composition comprising thedisclosed oligoribonucleotides, by any effective route. Exemplary routesof administration include, but are not limited to, injection (such assubcutaneous, intramuscular, intradermal, intraperitoneal, andintravenous), oral, sublingual, rectal, transdermal, intranasal, vaginaland inhalation routes.

Treating a subject can include any intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop, whether or not the subject has developed symptoms of thedisease. Ameliorating, with reference to a disease, pathologicalcondition or symptom refers to any observable beneficial effect of thetreatment. The beneficial effect can be evidenced, for example, by adelayed onset of clinical symptoms of the disease in a susceptiblesubject, a reduction in severity of some or all clinical symptoms of thedisease, a slower progression of the disease, a reduction in the numberof relapses of the disease, an improvement in the memory and/orcognitive function of the subject, a qualitative improvement in symptomsobserved by a clinician or reported by a patient, or by other parameterswell known in the art that are specific to viral infections generally orspecific viral infections.

A symptom can be any subjective evidence of disease or of a subject'scondition, for example, such evidence as perceived by the subject; anoticeable change in a subject's condition indicative of some bodily ormental state. A sign may be any abnormality indicative of disease,discoverable on examination or assessment of a subject. A sign isgenerally an objective indication of disease.

The administration of a pharmaceutical composition comprising thedisclosed oligoribonucleotides can be for prophylactic or therapeuticpurposes. When provided prophylactically, the treatments are provided inadvance of any clinical symptom of viral infection. Prophylacticadministration serves to prevent or ameliorate any subsequent diseaseprocess. When provided therapeutically, the compounds are provided at(or shortly after) the onset of a symptom of disease. For prophylacticand therapeutic purposes, the treatments can be administered to thesubject in a single bolus delivery, via continuous delivery (forexample, continuous transdermal, mucosal or intravenous delivery) overan extended time period, or in a repeated administration protocol (forexample, by an hourly, daily or weekly, repeated administrationprotocol). The therapeutically effective dosage of the treatments forviral infection can be provided as repeated doses within a prolongedprophylaxis or treatment regimen that will yield clinically significantresults to alleviate one or more symptoms or detectable conditionsassociated with viral infection.

In some examples of prophylactic administration of the pharmaceuticalcomposition, the pharmaceutical composition is administered as a vaccinecomprising one or more infectious disease specific antigens in order toinduce immunological memory against the one or more infectious diseaseorganisms from which the infectious disease specific antigens werederived. The immunological memory in turn provides the subject withimmunity from future infection from those infectious disease organismssuch that disease from those infectious disease organisms is preventedor lessened.

Suitable methods, materials, and examples used in the practice and/ortesting of embodiments of the disclosed invention are described below.Such methods and materials are illustrative only and are not intended tobe limiting. Other methods, materials, and examples similar orequivalent to those described herein can be used.

EXAMPLES

The following examples are illustrative of disclosed methods. In lightof this disclosure, those of skill in the art will recognize thatvariations of these examples and other examples of the disclosed methodwould be possible without undue experimentation.

Example 1—Materials and Methods

In Vitro Transcription and Gel Analysis:

RIG-I agonists were synthesized by designing complementary primers witha T7 promoter (Integrated DNA Technologies), annealing them, thensynthesizing with an in vitro transcription kit (Ambion) for 16 hours.RNA transcripts were DNase digested for 15 minutes at 37° C. thenpurified with an miRNeasy® kit (Qiagen). RNA was analyzed on adenaturing 15% TBE-urea polyacrylamide gel (Bio-Rad) following digestionwith 50 ng/μl of RNase A (Ambion) or 100 mU/μl of DNase I (Ambion) for30 minutes. Control wild type RNA is the dephosphorylated form of the WTsequence SEQ ID NO: 5 purchased from IDT.

Cell Culture, Transfections, and Luciferase Assays:

Lung epithelial A549 cells were grown in F12K (ATCC) supplemented with10% FBS (Access Cell Culture). Transfection of RNA and siRNA wereperformed with Lipofectamine RNAiMax® (Invitrogen) for 18-24 hours and48 hours, respectively. Poly (I:C) LMW was purchased from Invivogen. ForsiRNA knockdown, A549 cells were transfected with 30 pmol of human RIG-I(sc-61480), TLR3 (sc-36685), MDA5 (sc-61010), or control siRNA(sc-37007) (Santa Cruz Biotechnologies) using Lipofectamine RNAiMax®according to the manufacturer's guidelines. For luciferase assay, 200 ngIFN-β/pGL3 and 100 ng pRL-TK plasmids were co-transfected with 5′pppRNAusing Lipofectamine RNAiMax® for 24 h. Reporter gene activity wasmeasured by Dual-Luciferase Reporter Assay (Promega) according to themanufacturer's instructions. Relative luciferase activity was measuredas fold induction.

Monocyte Isolation and Differentiation into Monocyte-Derived DendriticCells:

Human peripheral blood mononuclear cells (PBMC) were isolated from buffycoats of healthy, seronegative volunteers in a study approved by the IRBand by the VGTI-FL Institutional Biosafety Committee (2011-6-JH1).Written informed consent approved by the VGTI-FL Inc. ethics reviewboard (FWA#161) was provided to study participants. Research conformedto ethical guidelines established by the ethics committee of the OHSUVGTI and Martin Health System. Briefly, PBMC were isolated from freshlycollected blood using the Ficoll-Paque™ PLUS medium (GE Healthcare Bio)as per manufacturer's instructions. CD14⁺ monocytes were isolated bypositive selection using CD14 microbeads and a magnetic cells separatoras per kit instructions (Miltenyi Biotech). Purified CD14⁺ monocyteswere cultured for 7 days in 100 mm dishes (15×10⁶ cells) in 10 mL ofcomplete monocyte differentiation medium (Miltenyi Biotech). On day 3,the medium was replenished with fresh medium.

Quantitative Real-Time RT-PCR:

Total RNA was isolated from cells using an RNeasy® kit (Qiagen)according to the manufacturer's instructions. RNA was reversetranscribed using the SuperScript® VILO cDNA synthesis kit (Invitrogen)according to the manufacturer's instructions. PCR primers were designedusing Roche's Universal Probe Library Assay Design Center(www.universalprobelibrary.com.) Quantitative RT-PCR was performed on aLightCycler® 480 Probes Master (Roche.) All data are presented as arelative quantification with efficiency correction based on the relativeexpression of target gene versus GAPDH as the invariant control. Primersand probes are described in the attached Sequence Listing.

Immunoblot Analyses:

Whole cell extracts were separated in 4-20% acrylamide Mini-Protean® TGXprecast gels (Bio-Rad) by SDS-PAGE and transferred to anImmobilon-P^(SQ) PVDF membrane (Millipore) for 1 hour at 100V in abuffer containing 30 mM Tris, 200 mM glycine and 20% methanol. Membraneswere blocked for 1 hour at room temperature in blocking buffer (Odyssey)then probed with the following primary antibodies: anti-RIG-I (EMDMillipore), anti-IFIT1 (Thermo Fisher Scientific), anti-pSTAT1 (CellSignaling), anti-STAT1 (Cell Signaling), anti-pIRF3 S396 (CellSignaling), anti-IRF3 (Cell Signaling), anti-β-actin (Odyssey), oranti-NS1. Antibody signals were detected by immunofluorescence using theIRDye® 800CW and IRDye® 680RD secondary antibodies (Odyssey) and theLI-COR imager (Odyssey.)

Flow Cytometry Analysis:

Percentage of dengue-infected cells was determined by standardintracellular staining (ICS) using a mouse IgG2a mAb, specific fordengue E protein (clone 4G2) followed by staining with a secondaryanti-mouse antibody coupled to PE (Biolegend). Prior to surface stainingof dendritic cell maturation markers, MDDCs were stained for 5 minuteswith human TruStain FcX (Biolegend) followed by staining with CD83-PE(Biolegend), CD86-Pacific Blue (Biolegend), or CCR7-PE Cy5 (Biolegend)for 15 minutes at 4° C. Cells were analyzed on an LSRII® flow cytometer(Becton Dickinson). Calculations and population analyses were done usingFACS Diva software.

Virus Production and Infection:

Dengue serotype 2 strain New Guinea C (NGC) was used to infect confluentmonolayers of C6/36 insect cells at an MOI of 0.5. Virus was allowed toadsorb for 1 hour at 28° C. in serum-free DMEM. Serum-free DMEM was usedto wash the monolayer then replaced with DMEM/2% FBS. After 7 days ofinfection, the medium was harvested, cleared by centrifugation (1100 g,10 min), and the supernatant was concentrated by centrifugation (1100 g)through a 15 ml Amicon® Centrifugal Filter Unit (Millipore). The viruswas concentrated by ultracentrifugation on a sucrose density gradient(20% sucrose cushion) using a Sorvall® WX 100 Ultracentrifuge(ThermoScientific) for 2 hours at 134,000 g, 10° C. and the brake turnedoff. Concentrated virus was then washed to remove sucrose using a 15 mlAmicon® tube. After 2 washes, the virus was resuspended in DMEM/0.1%BSA. Titers of dengue stocks were determined by FACS, after infectingVero cells by immunofluorescent staining of intracellular dengue Eprotein 24 hours post infection. For dengue challenge experiments, A549cells were infected using dengue at an MOI of 0.5 in serum free F12K for1 hour at 37° C. Medium was replaced with complete medium for 24 h priorto analysis. All procedures with live dengue were performed in abiosafety level 2+ facility at the Vaccine and Gene TherapyInstitute-Florida.

Influenza H1N1 strain A/Puerto Rico/8/34 was amplified in Madin-Darbycanine kidney (MDCK) cells and virus titer was determined by standardplaque assay. Cells were infected in 1 ml medium without FBS for 1 hourat 37° C. Inoculum was aspirated and cells were incubated with completemedium for 24 hours, prior to analysis. For viral infections,supernatants containing soluble factors induced following 5′pppRNAtreatment was removed and kept aside during infection. Cells were washedonce with PBS and infected in a small volume of medium without FBS for 1h at 37° C.; then supernatant was added back for the indicated period oftime. Procedures with live influenza H1N1 strain A/Puerto Rico/8/34 wereperformed at McGill University in a biosafety level 2+ facility.

Influenza reassortant H5N1 virus (H5N1-PR8) was generated usinghemagglutinin (HA) and neuraminidase (NA) genes were derived from theH5N1 virus (HA from influenza A/Vietnam/1203/2004 and NA from influenzaA/Thailand/1(KAN-1)/2004). The internal viral proteins were derived fromthe A/Puerto Rico/8/1934 (PR8) mouse adapted influenza A virus. Thepropagation of the H5N1-PR8 reassortant viruses was performed using MDCKcells. Anesthetized female BALB/c mice were infected intranasally withthe lethal dose (5×10³ PFU in 50 μL PBS) of the H5N1-PR8. All procedureswith influenza reassortant H5N1-PR8 were performed in a biosafety level2+ facility at the Vaccine and Gene Therapy Institute-Florida.

Virus Like Particle (VLP) Vaccine:

The H5N1-PR8 VLP were purified from HEK 293 T cells which weretransfected using Lipofectamine2000® (Invitrogen) with 5 μg of eachplasmid DNA expressing H5N1 A/Vietnam/1203/2004 HA and H5N1A/Thailand/1(KAN-1)/2004 NA (codon optimized); and 10 μg of plasmid DNAexpressing HIV gag. Cells were incubated for 72 h at 37° C. andsupernatants containing VLPs were collected, sterile filtered and VLPwere purified by centrifugation at 100,000×g through a 20% glycerolcushion and resuspended in PBS. Total protein was quantified using BCAprotein assay (Thermo Fisher Scientific) and VLPs were aliquoted in PBSand stored at −80° C.

Sickness Score:

The sickness score was generated by evaluation of animal activity,hunched back, and ruffled fur. The final score was the addition of eachindividual score resulting in the minimum score 0 for a healthy mouseand 1-4 for a sick mouse.

In Vivo Administration of 5′pppRNA and Influenza Infection Model:

BALB/c mice (6-8 weeks of age, Jackson Laboratories) were housed in cageunits, fed ad libitum, and cared for under USDA guidelines forlaboratory animals. For vaccination, mice were anesthetized with IsoSol®(Patterson Veterinary) and vaccinated via the intramuscular route with 3μg (based on HA content) of purified VLP (in 50 μl PBS) with or without25 μg 5′pppRNA as adjuvant and then challenged or boosted with the samedose at week 3 with the identical vaccine formulation. The 5′pppRNA wascomplexed with in vivo-jetPEI® (PolyPlus, France) at an N/P ratio of 8according to the manufacturer instructions. Animals were monitored forsurvival and morbidity (weight loss, ruffling fur, hunched back,lethargy) weekly during the vaccination regimen and each day during theviral challenge. Blood samples for serological analysis were collectedfrom anesthetized mice via retro-orbital sinus. Blood was allowed toclot at room temperature and sera was removed and frozen at −80° C.after centrifugation. All procedures were in accordance with the NRCguide for the Care and Use of Laboratory Animals and the Animal WelfareAct.

Hemagglutination Inhibition Activity:

The hemagglutination inhibition (HAI) assay was used to assessfunctional antibodies to the HA able to inhibit agglutination of horsered blood cells. The procedure was adapted from the Center for DiseaseControl influenza surveillance manual and performed as previouslydescribed.

Serological Assays:

A quantitative ELISA was performed to assess anti-HA specific IgG inimmune serum. Purified rHA (25 ng) was used to coat each well of a96-well plate. Plates were blocked (25° C. for 2 h) with blocking buffer(PBS pH 7.5 containing 0.05% Tween 20, 5% BSA Fraction V, and 2% bovinegelatin) and then incubated with serial dilutions of each serum sample(37° C. for >90 min). After five washes with PBS, samples were incubated(37° C. for >90 min) with horseradish peroxidase rabbit anti-mouse IgG(1:2500) diluted in blocking buffer. The unbound antibody was removed,and the wells were washed five times with PBS. Samples were thenincubated (10-20 min) with2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) substrateand the colorimetric change was measured as the optical density (O.D. at414 nm) by a spectrophotometer (BioTek, Winooski, Vt., USA). The O.D.value of the sample-matched BSA− only coated plates was subtracted fromthe O.D of samples where plates were coated with BSA+rHA.

Histology:

Lungs were harvested and prepared for immunohistochemistry usingstandard procedures. After euthanasia, chest cavity was opened and thelungs were gently inflated intratracheally with 4° C. 4%paraformaldehyde in PBS, removed and immersed in 4% paraformaldehyde at4° C. overnight. The next day, the solution was replaced with 70%ethanol and tissues were kept at 4° C. for up to 2 weeks. Tissuesections were embedded in paraffin, sectioned into slices (^(˜)5 μm inthickness), and stained with hematoxylin and eosin (H&E) or leftunstained. Tissue sections were imaged with a Qimaging Micropublisher®5.0 RTV digital camera on an Olympus BX61 fluorescence microscope. Toquantify the number of apoptotic lung cells, representative sectionswere deparaffinized and rehydrated in xylene and graded alcohols,respectively, using standard procedures, and TUNEL assay was performedaccording to manufacturer's instructions (Roche, Mannheim, Germany). Thepercentages of TUNEL-positive cells within the tissue sections weredetermined by counting at least 100 cells each from eight randomlyselected fields.

In Vivo Administration of 5′pppRNA and Chikungunya Infection Model:

Control, WT, M5, or M8 5′pppRNA were administered to adult mice using aprotocol similar to that of the influenza infection in vivo model. Micewere injected intramuscularly with 2 or 10 μg RNA oligonucleotides incombination with in vivo JetPEI for 24 hours then infected withchikungunya via footpad injection. Treatments were assessed via caliperfor footpad swelling or viral RNA copy number via qPCR using chikungunyavirus-specific primers. All procedures with chikungunya virus wereperformed at Oregon Health and Science University.

Example 2—RIG-I Agonists Comprising Mutations Away from the 5′ and 3′Termini of Vesicular Stomatitis Virus Sequence have Improved Antiviraland Anti-Inflammatory Properties Relative to Wild Type

RIG-I agonists derived from the 5′ and 3′ termini of vesicularstomatitis virus (which is referred to as WT, WT-VSV, or 5′pppSEQ ID NO:5 herein) were tested for antiviral and inflammatory properties. Severalmodified forms of oligoribonucleotides comprising a 5′triphosphosphatewere synthesized by in vitro transcription and tested for biologicalactivity. Several sequences displayed increased activity; however, theM5 (5′pppSEQ ID NO: 10) and M8 (5′pppSEQ ID NO: 13) 5′-triphosphateoligoribonucleotides resulted in antiviral and immune cytokine levels10-100 times higher than WT (FIG. 1). The activity of M8 was alsosuperior to that of TLR3 activator poly (I:C) or RIG-I agonist CL9, aSELEX-selected aptamer sequence.

The first generation of sequences, M1, M2, M3, M4, and M5, (5′pppSEQ IDNO: 6, 5′pppSEQ ID NO: 7, 5′pppSEQ ID NO: 8, 5′pppSEQ ID NO: 9, 5′pppSEQID NO: 10 respectively) were synthesized by in vitro transcription, andanalyzed for single products (FIG. 2A) that were sensitive to RNase Abut not DNase I digestion (FIGS. 2B and 2C), indicating that the invitro transcribed products were RNA. RIG-I agonists were then assessedfor IFN-β activation, cytokine stimulation, as well as virus inhibitoryactivity (FIG. 3). In all cases, M5 was the best candidate of the group,as it activated IFN-β at lower concentrations (FIG. 3A), stimulatedantiviral and innate immune response genes—ISG56, IFNβ, and IL-1a tohigher levels than wtVSV (FIG. 3B), and effectively blocked influenza(FIG. 3C) and dengue (FIGS. 3D and 3E) replication in lung epithelialcells, when compared to other RNA agonists. Other agonists were able tostimulate antiviral IFNβ production and block virus replication, butactivity was approximately 10-fold lower than M5, indicating theimproved activity of this novel sequence.

A second generation of RNA sequences based on the structure of M5revealed that increasing the double stranded RNA length improvedantiviral activity (FIG. 4). M6 (5′pppSEQ ID NO: 11) and M7 (5′pppSEQ IDNO: 12) were similar to M5 in their ability to block dengue infection,as seen by the percentage of cells positive for dengue E protein (FIG.4A) and at the RNA level (FIG. 4B). M8 (5′pppSEQ ID NO: 13), however, atcomparable concentrations, completely inhibited dengue replication atboth levels (FIGS. 4A and 48). This was further tested by lowering theconcentrations used in the dengue challenge experiment and even at aconcentration as low as 0.01 ng/ml, no dengue-positive cells weredetected in M8-treated cells (FIG. 4C). In all previous experiments, noRNA ribonucleotide was able to elicit the tremendous viral inhibition atsuch a low concentration.

A comparison of the original WT 5′-triphosphate oligoribonucleotide(5′pppSEQ ID NO: 5) to the M5 and M8 sequences reveal that although the5′-triphosphate tail and portions of the double stranded region remainintact (FIG. 5), structure and sequence are altered, indicating theirimportance in antiviral and inflammatory activity.

To ensure that M8 acts as a RIG-I agonist, cells were knocked down forthe RIG-I sensor, or TLR3/MDA5 sensors before treatment with M8. Upon M8treatment and dengue infection of cells, the absence of RIG-I renderedthe M8-treated cells sensitive to virus replication (FIG. 6A), whereassilencing TLR3 and MDA5 (two distinct RNA sensors) did not affect M8function.

This result demonstrates that M8 activity signals through the RIG-Ipathway. This result is further supported by the fact that RIG-Idepleted cells treated with M8 were unable to upregulate the expressionof STAT-1, a component of the IFN antiviral pathway, whereas in theabsence of TLR3 and MDA5 sensors, M5-treated cells stimulated theantiviral pathway (FIG. 6B).

WT, M5, and M8 RNA were assessed for their ability to inhibit differentviruses and/or different disease models. In each assay, M8 was moreactive than WT and M5. Influenza (FIG. 7A) and dengue viruses (FIG. 7B)in A549 and human immune cells respectively were inhibited by M8 atlower concentrations than WT or M5. In monocyte-derived dendritic cells,M8 induced dendritic cell maturation as effectively as the positivecontrol LPS (FIG. 7C); the increase in surface markers CD86 and CD83illustrate the maturation of DC. This ability of M8 to drive dendriticcell maturation indicates that this RNA sequence can not only stimulateinnate antiviral capacity, but also increase the antigen presentingcapacity of DC, a function that stimulates the transition from innate toadaptive cellular immunity.

Further characterization of the RNA oligonucleotides led to redesign ofthe M8 sequence (FIG. 8). The sequence length was maintained at 99nucleotides; however, half of the sequence was modified with alternatingAAU (adenine, adenine, uracil) bases (M8A-5′pppSEQ ID NO: 14) or theentire sequence was randomized (M8B-5′pppSEQ ID NO: 15). Upon viralchallenge, M8 performed better than its two variants, although M8A stillsignificantly inhibited dengue replication (FIG. 9A). M8B blocked virusat a higher concentration of 1 ng/ml. The enhanced activity of M8 versusM8A and M8B was also evident in the expression of several cytokines andantiviral genes (FIG. 9B), suggesting that primary RNA sequence alsoinfluences the activity of the RNA agonist.

The novel sequences M5 and M8 were then evaluated in vivo for theirantiviral and adjuvant activities. To assess the capacity of M5 toincrease antibody responses to influenza virus like particle (VLP)vaccination, Balb/c mice were injected intramuscularly (IM) with 25 μgof M5, 3 mg VLP (based on haemagglutinin (HA) content) or thecombination of VLP-M5 (FIG. 10). Three weeks after the firstvaccination, a same-dose booster vaccine was injected. Two weeks afterthe second dose, mice were bled and serum was isolated to determinetotal HA-specific IgG antibody titers. Addition of M5 increasedHA-specific (FIGS. 11A and 118) as well as neutralizing antibody titers(FIG. 11C), as determined by HA-specific ELISA and haemagglutinationinhibition assays (HAI), respectively. Mice were then challenged with alethal dose of the reassorted H5N1 virus (5,000 plaque formingunits/animal) intranasally, and their weight, survival, and sicknessscore were assessed over three weeks. Control and M5-vaccinated micecontinuously losing weight and were humanely euthanized at nine dayspost-challenge (FIG. 11D). Mice vaccinated only with VLPs lost weight,displayed mild signs of sickness, and two mice had to be euthanized atday 16 (FIGS. 12A and 12B). Only mice vaccinated with VLP+M5continuously gained weight and displayed no overt signs of infection.Post-mortem examination of lung tissues revealed increased apoptoticcell death (TUNEL-positive cells) in the control group and in micevaccinated with M5 (FIG. 12C). Additionally, H&E staining revealedincreased inflammation in the bronchial airways in control and in micevaccinated with M5 (FIG. 13, yellow arrow).

To evaluate the activity of M5 and M8 with a different route ofadministration, mice were injected intramuscularly (as with a vaccine)with of WT, M5 or M8 and the induction of IFNβ mRNA levels afterinjection were measured. At indicated time points, muscles wereharvested, RNA isolated, and the levels of IFNβ were determined byRT-PCR (FIGS. 14A and 14B). Based on these results, M8 induces 3-5 foldhigher levels of IFNβ mRNA 6 h post-injection.

In vivo antiviral activity of M5 and M8 sequences was also examined in amurine model of chikungunya virus pathogenesis, in which swelling of thefootpad is used as a marker of inflammation and arthritis. Both 2 and 10μg of either M5 or M8 were efficient in preventing swelling in theipsilateral foot pad (FIG. 15A). M5 and M8 also inhibited chikungunyaviremia (virus in the blood), as assessed by measuring chikungunya viralRNA levels in the blood (FIG. 15B).

Example 3—Sequence Specific Modifications Enhance the Broad-SpectrumAntiviral Response Activated by RIG-I Agonists

The cytosolic RIG-I (retinoic acid-inducible gene I) receptor plays apivotal role in the initiation of the immune response against RNA virusinfection by recognizing short 5′-triphosphate (5′ppp)-containing viralRNA and activating the host antiviral innate response. Disclosed hereinare novel 5′ppp RIG-I agonists of various lengths, structures, andsequences. The generation of the antiviral and inflammatory responses inhuman epithelial A549 cells, human innate immune primary cells, andmurine models of influenza and chikungunya viral pathogenesis wereevaluated. A 99-nucleotide, uridine-rich hairpin 5′-pppRNA termed M8stimulated an extensive and robust interferon response compared to othermodified 5′-pppRNA structures, RIG-I aptamers, or poly(I-C).Manipulation of the primary RNA sequence alone was sufficient tomodulate antiviral activity and inflammatory response. These mechanismswere dependent exclusively on RIG-I and independent of MDA5 and TLR3.Both prophylactic and therapeutic administration of M8 effectivelyinhibited influenza virus and dengue virus replication in vitro.Furthermore, multiple strains of influenza virus that were resistant tooseltamivir, an FDA-approved therapeutic treatment for influenza, werehighly sensitive to inhibition by M8. Finally, prophylactic M8 treatmentin vivo prolonged survival and reduced lung viral titers of micechallenged with influenza virus, as well as reducing chikungunyavirus-associated foot swelling and viral load. Altogether, these resultsdemonstrate that 5′-pppRNA can be rationally designed to achieve amaximal RIG-I-mediated protective antiviral response againsthuman-pathogenic RNA viruses.

The development of novel therapeutics to treat human-pathogenic RNAviral infections is an important goal to reduce spread of infection andto improve human health and safety. Disclosed herein is an RNA agonistwith enhanced antiviral and inflammatory properties against influenza,dengue, and chikungunya viruses. A novel, sequence-dependent,uridine-rich RIG-I agonist generated a protective antiviral response invitro and in vivo and was effective at concentrations 100-fold lowerthan prototype sequences or other RNA agonists, highlighting the robustactivity and potential clinical use of the 5′-pppRNA against RNA virusinfection. Altogether, the results identify a novel, sequence-specificRIG-I agonist as an attractive therapeutic candidate for the treatmentof a broad range of RNA viruses, a pressing issue in which a need fornew and more effective options.

Human-pathogenic RNA viral infections, including influenza, dengue, andchikungunya, pose significant threats to human health and safety. Forthis reason, the development of prophylactic and therapeutic antiviralsto treat and limit spread of infection remains a growing unmet medicalneed. Currently, there are no therapeutics for the prevention ortreatment of dengue or chikungunya infections, and approved antiviralcompounds to treat influenza have significant problems associated withtheir use. For instance, anti-influenza agents such as amantadine andrimantadine block virus uncoating but are not recommended for currentlycirculating influenza A or B virus strains because of widespreadresistance (Drinka P J and Haupt T, J Am Geriatr Soc 55, 923-926 (2007);incorporated by reference herein). Oseltamivir, a neuraminidaseinhibitor, is also active against influenza A and B viruses at earlystages of infection but has given rise to drug-resistant mutants (BloomJ D et al, Science 328, 1272-1275 (2010); incorporated by referenceherein). Therapies that harness and activate the natural immune defensemay circumvent the issues of the emergence of drug resistance andoff-target effects. The innate immune system provides the initialbarrier against viral infection, initiating a cascade of signalingpathways and sensors that detect and clear the intruding virus. RNAviruses possess pathogen-associated molecular patterns (PAMPs) that aresensed by pattern recognition receptors (PRR) (Brennan K and Bowie A G,Curr Opin Microbiol 13, 503-507 (2010); Tekeuchi O and Akira S, ImmunolRev 227, 75-86 (2009); Wilkins C and Gale Jr. M, Curr Opin Immunol 22,41-47 (2010); Yoneyama M and Fujita T, Rev Med Virol 20, 4-22 (2010);and Akira S et al, Cell 124, 783-801 (2006); all of which areincorporated by reference herein). Toll-like receptor (TLR) and RIG-I(retinoic acid-inducible gene I)-like receptor (RLR) families generatean innate immune response upon recognition of broadly conserved PAMPs onviruses and bacteria Goubau D et al, Immunity 38, 855-860 (2013);incorporated by reference herein). RIG-I recognizes shortdouble-stranded RNA (dsRNA) oligonucleotides of <100 nucleotides inlength bearing 5′-triphosphate or 5′-diphosphate termini (Goubau D etal, Nature 514, 372-375 (2014); incorporated by reference herein), whileMDA5 generally recognizes longer, dsRNA (>300 nucleotides) lacking a5′-triphosphate moiety. RIG-I detects viral RNA through its helicasedomain (Bamming D and Horvath C M, J Biol Chem 284, 9700-9712 (2009);Fujita T et al, Biochimie 89, 754-760 (2007); Jiang X et al, Immunity36, 959-973 (2012); and Schmidt A et al, J Mol Med (Berl) 80, 5-12(2011); all of which are incorporated by reference herein), leading toconformational changes that expose the effector caspase activation andrecruitment domain (CARD), which in turn interacts with themitochondrial adaptor MAVS (Kawai T et al, Nat Immunol 6, 981-988(2005); Komuro A et al, Cytokine 43, 350-358 (2008); and Meylan E et al,Nature 437, 1167-1172 (2005); all of which are incorporated by referenceherein). MAVS serves as a signaling platform for protein complexes thattrigger activation of the transcription factors NF-κB, interferon(IFN)-regulatory factor 3 (IRF-3), and IFN regulatory factor 7 (IRF-7),leading to the induction of antiviral programs that include productionof type I IFN as well as proinflammatory cytokines and antiviral factors(Belgnaoui S M et al, Curr Opin Immunol 23, 564-572 (2011); Kawai T andAkira S, Ann NY Acad Sci 1143, 1-20 (2008); Pichlmair A et al, Science314, 997-1001 (2006); Rehwinkel J and Reis e Sousa C, Science 327,284-286 (2010); Rehwinkel J et al, Cell 140, 397-408 (2010); andTakeuchi O and Akira S, Cell 140, 805-820 (2010); all of which areincorporated by reference herein). A secondary response is induced byIFN binding to the type I IFN receptors (IFN-α/BR), which activate theJAK-STAT pathway and induce interferon-stimulated genes (ISGs) and theantiviral immune response (Sadler A J and Williams B R, Nat Rev Immunol8, 559-568 (2008) and Schoggins J W et al, Nature 472, 481-485 (2011);both of which are incorporated by reference herein). More recently,RIG-I has been shown to function as both an innate sensor and anantiviral factor by triggering downstream interferon signaling eventsand disrupting the interaction between hepatitis B virus (HBV)polymerase and pgRNA (Sato S et al, Immunity 42, 123-132 (2015);incorporated by reference herein). Overall, novel therapies specificallytargeting the RIG-I pathway have the potential to elicit abroad-spectrum, antiviral, inflammatory, and immune modulatory responseand thus represent an attractive strategy for the design and developmentof novel and improved antiviral therapies.

The antiviral activity of a short in vitro synthesized 5′-triphosphateRNA (5′-pppRNA) derived from the 5′ and 3′ untranslated regions (UTRs)of the vesicular stomatitis virus (VSV) genome (termed wild-type [WT]5′-pppRNA) herein has been disclosed (Goulet M L et al, PLoS Pathog 9,e1003298 (2013); incorporated by reference herein). Pretreatment with WT5′-pppRNA activated the RIG-I signaling pathway and triggered a robustantiviral response that significantly decreased infection by severalpathogenic viruses, including dengue virus, hepatitis C virus (HCV),H1N1 influenza virus A/PR/8/34, and HIV-1. In vivo, intravenous deliveryof the WT 5′-pppRNA stimulated an antiviral state that inhibited a broadspectrum of RNA viruses and protected mice from lethal influenza viruschallenge (Olagnier D et al, J Virol 88, 4180-4194 (2014); incorporatedby reference herein).

The nature of the ligand recognized by RIG-I has been the subject ofnumerous studies. Structural motifs, lengths, and sequences ofvirus-derived 5′-pppRNA and other RNA agonists have been analyzed andfound to play critical roles in the response to viral infection. Invitro-synthesized RNA with a 5′-terminal triphosphate moiety was firstidentified as a RIG-I agonist (Hornung V et al, Science 314, 994-997(2006) and Schlee M et al, Immunity 31, 25-34 (2009); both of which areincorporated by reference herein) however, more recently, it wasdiscovered that a 5′-diphosphate terminus could also be recognized byRIG-I to mediate an antiviral response (Goubau et al, 2014 supra). RIG-Istimulation was dependent on double stranded RNA at least 20 nucleotideslong possessing blunt base pairing at the 5′ end Schlee M and HartmannG, Mol Ther 18, 1254-1262 (2010); incorporated by reference herein).Ligands recognized by RIG-I include double-stranded RNA, found inconserved 5′ and 3′ UTRs of negative single-strand RNA virus genomes,displaying high base pair complementarity and a panhandle structure.

Most studies on RIG-I antiviral properties have used virus derived5′-pppRNA or defective interfering particles or commercially availablesynthetic 5′-pppRNA to trigger the RIG-I antiviral response. In thepresent study, we report for the first time the sequence-specificactivity of a novel RIG-I agonist active against a number of RNA virusesboth in vitro and in vivo. Modifications to the structure, length, andsequence of a prototypical 5′-triphosphate-containing RNA significantlypotentiated the host antiviral response against influenza, dengue andchikungunya virus infections while maintaining the specificity forinteraction with the RIG-I cytosolic sensor.

5′-pppRNA Sequence and Structure Determine Antiviral Activity:

Optimization of the prototypical VSV-derived WT 5′-pppRNA (Goulet et al,2013 supra and Schlee et al, 2009 supra) resulted in novel structureswith enhanced antiviral activity compared to the short form ofpolyinosinic-poly(C) [poly(I⋅C)], a well-known and -characterized TLR3agonist, or the SELEX-selected RIG-I aptamers CL2 and CL9 Hwang S Y etal, Nucleic Acids Res 40, 2724-2733 (2012); incorporated by referenceherein). A representation of the predicted secondary structure of eachof the agonists generated is included in FIG. 16A. The 5′-triphosphateRNA agonists were generated by in vitro transcription as previouslydescribed (Goulet et al 2013 supra), and gel analysis revealed a singleproduct (FIG. 16B) susceptible to digestion by RNase A but not by DNaseI. In the first generation of modified sequences, modifications to theprimary RNA sequence that eliminated mismatch in the double-stranded RNA(dsRNA) region (M1), removed the panhandle sequence (M2), introducedadditional bases to the dsRNA region (M3), or generated a short dsRNApin loop (M4) did not enhance agonist activity when assayed for IFN-γinduction or antiviral effects. However, the addition of extra basepairs to the M4 sequence resulted in a 59-nucleotide dsRNA structure(M5) that induced higher levels of IFN-γ and ISG56 and inhibitedinfluenza virus and dengue virus (DenV) replication more effectivelythan the prototypical WT 5′-pppRNA. Further modification of M5, via theaddition of AU base pairs to increase the length of the dsRNA stemstructure (M6 to M8), further enhanced antiviral activity. Human lungepithelial A549 cells treated with M8 were protected from DenV infectionat concentrations ^(˜)2-log lower than those treated with M5. Theenhanced antiviral activity of M5 and M8 was increased 10- and 100-fold,respectively, compared to WT, as measured by antiviral activity andstimulation of innate immune molecules (FIGS. 16C to 16G).

To examine the relative activities of the optimized agonist, A549 cellswere transfected with WT, M5, or M8 5′-pppRNAs, and expression ofvarious cytokines and IFN-stimulated genes (ISG) was evaluated byquantitative PCR; equimolar amounts of RNA were used to compensate forthe disparities in agonist length. Expression of theinterferon-stimulated gene ISG56, type I and III interferons (IFN-γ andinterleukin 29 [IL-29]), and the cytokine tumor necrosis factor alpha(TNF-α), measured at the RNA level, was significantly higher inM8-treated cells than in cells treated with other agonists (FIG. 16C).ISG56, IFN-γ, TNF-α, and IL-29 mRNA expression levels in M8-treatedcells were 33.7-, 47.9-, 1,397.3-, and 185.0-fold higher than those inWT-treated cells, respectively. Increased mRNA expression levels wereaccompanied by enhanced interferon-stimulated response element (ISRE)and IFN-γ activity in a luciferase reporter assay (FIG. 16D) and byincreased phosphorylation of IRF3 at 5396 and expression of ISG56,STAT1, and RIG-I protein (FIG. 16E) in M8-treated cells compared to WT-or M5-treated cells. At low (0.01-ng/ml) concentrations of M8, A549cells were completely protected from influenza virus infection, whereascells treated with WT, M5, or CL9 aptamer displayed detectable NS1protein expression at 24 h after infection (FIG. 16F). Similarly, cellstreated with M8 and then challenged with DenV were completely protectedagainst infection (FIG. 16G). Production of IFN-γ within the supernatantof 5′-pppRNA-treated cells was 17.8-fold greater in M8-treated samplesthan in the WT, as measured by enzyme-linked immunosorbent assay(ELISA), and transfer of the supernatant from M8-treated A549 cells ontofreshly seeded cells 24 h later inhibited dengue replication asefficiently as direct treatment of M8, suggesting that antiviralcytokine production is associated with the enhanced antiviral propertiesof M8. The 5′-triphosphate moiety was required to generate the antiviralresponse. In addition, M8 was not cytotoxic to A549 cells, since highconcentrations of agonist did not reduce cell viability. Altogether,these results identify a 5′-pppRNA RIG-I agonist with enhanced antiviralactivity against influenza virus and dengue virus infections.

Antiviral Activity is Dependent on RNA Structure:

The enhanced activity of M8 appeared to be the consequence ofmodification of sequence, length, and ultimately structure of the RNA.To examine whether sequence alone was sufficient to alter the antiviraland inflammatory activity, new sequence modifications that maintainedthe 99-nt looped structure were introduced into the M8 sequence (FIG.17A). Substitution of the poly(U) stretch with alternating AAUs (M8A) orACs (M8C), randomization of the entire 99-nt sequence (M8B), andrandomization of the WT derived region while leaving the poly(U) stretchintact (M8D) all resulted in varied effectiveness. Those with no (M8D)or minor (M8A) changes to the poly(U) stretch maintained theinducibility of ISGs, as exemplified by increased expression ofchemokines, cytokines (CXCL10, IL-6, IL-1α, and TNF-α), and inflammatoryand antiviral mRNA (ISG56 and IFN-γ) (FIG. 17B). ISRE and IFN-γ relativeactivities of M8B and M8D measured by luciferase assay were comparableto those of the original M8 sequence (FIG. 17C). Although all variantswere capable of inducing expression of ISG56, STAT1, and RIG-I, M8C hadthe lowest activity (FIG. 17D). Sequence modification alone wassufficient to alter viral inhibition properties (FIG. 17E). While M8,M8A, and M8D at 0.01 ng/ml completely abrogated DenV replication, thenumber of DenV positive cells was reduced to 35% and 70%, respectively,when M8B and M8C were used as RIG-I agonists. Further sequencemodifications, including insertion of a CCC motif to the poly(U) region,replacement of the poly(U) region with alternating AAC, or introductionof AAAAA segments throughout the sequence, resulted in decreasedantiviral and inflammatory activity (data not shown), thus demonstratingthat the poly(U) moiety was critical for M8 activity.

M8 Activity is Dependent on Functional RIG-I Signaling:

To ensure that the novel RNA agonist maintained specificity for theRIG-I pathway, siRNA directed against various RNA sensors was used toknock down RIG-I, TLR3, and MDA5, respectively. While MDA5 and TLR3knockdown did not affect M8-mediated antiviral activity (FIG. 18A) asdetermined by immunoblot analysis of several ISGs, cells knocked downfor RIG-I failed to induce a ISG56 or STAT1 response compared to siRNAcontrol-treated cells. Additionally, RIG-I knockdown abolished TLR3 andMDA5 expression, suggesting an upstream regulatory activity of RIG-I onother pattern recognition receptors. In the absence of RIG-I, M8 wasunable to induce secretion of type I and III interferon, CXCL10, orTNF-α, whereas MDA5 or TLR3 knockdown did not block antiviral andinflammatory activities (FIG. 18B). Moreover, RIG-I was required forM8-induced antiviral activity against influenza virus and dengue virusreplication (FIGS. 18C to 18F), as determined by the diminishedexpression of influenza NS1 viral protein (FIG. 18C) and decreased DenVtiter (FIG. 18D) in cells lacking RIG-I. The absence of RIG-I alsoresulted in an increase in viral protein compared to influenzavirus-infected control cells, indicating that the loss of the RIG-Isensor enhanced viral replication. Additionally, to rule out thepossibility that the poly(U)-rich moiety of M8 was recognized by TLR7and TLR8, known targets of single stranded poly(U)-RNA molecules(Diebold S S et al, Science 303, 1529-1531 (2004); incorporated byreference herein), expression of these receptors was blocked. M8 wasable to block DenV infection—based on evaluation of DenV 2E proteinexpression by ICS staining and by quantification of viral RNA (FIGS. 3Eand F)—despite the absence of TLR7 and TLR8, Altogether, these dataindicate that the M8 5′-pppRNA antiviral activity is RIG-I specific.

M8 Activates Greater Breadth and Intensity of Innate Immune Response:

The response elicited by the optimized M8 RIG-I agonist was examinedusing monocyte-derived dendritic cells (Mo-DCs) transfected with RIG-Iagonists at equimolar doses and examined induction of antiviral andinflammatory genes and inhibition of viral replication. Using acustomized high-throughput BioMark chip, nearly all genes selected in apanel of antiviral and inflammatory genes, including type I interferons(IFNB1), type III interferons (IL28RA and IL-29), proinflammatorycytokines and chemokines (CCL5, IL1B, and IL-6), andinterferon-stimulated genes (DDX56, IFIT1, ISG15, and MX1), wereupregulated by M8 (FIG. 19A). Selected genes from the BioMark chip arerepresented in FIG. 19B. Induction of inflammatory markers wasaccompanied by an increase in pSTAT1, as measured by flow cytometry(geomean fluorescence [P=0.03]) (FIG. 19C). M8 inhibited influenza virusreplication in Mo-DCs, as exhibited by a decrease in NS1 protein (FIG.19D) and a 2-log-fold decrease in viral titer (FIG. 19E), recapitulatingthe results observed in A549 cells. Dengue viral replication wascompletely inhibited in M8-treated cells, whereas WT and M5 at the sameconcentration reduced but did not completely eliminate DenV E proteinexpression (FIG. 19F). Increased expression of STAT1, RIG-I, and ISG56in M8-treated Mo-DCs accompanied the inhibition of dengue viral proteinexpression (FIG. 4F). Altogether, these results emphasize that both theintensity and the breadth of antiviral gene expression were increaseddramatically in M8-treated primary human DCs, compared to treatment byother RNA agonists.

M8 Possesses Enhanced Antiviral Activity Against Multiple Strains ofInfluenza Virus Compared to Oseltamivir:

As shown in FIG. 16F, M8 5′-pppRNA efficiently blocked H1N1 influenzavirus A/Puerto Rico/8/1934 at concentrations 100-fold and 10-fold lowerthan those of the WT and M5, respectively. The capacity of the M8agonist to inhibit three different pandemic and seasonal influenza Avirus strains (H1N1 A/Brisbane/59/2007, H3N2 A/Brisbane/10/2007, andH5N1-PR/8 reassortant) in addition to H1N1/A/PR/8/1934, was evaluated.Some of the strains are resistant to oseltamivir, an FDA-approvedantiviral drug for influenza virus and the current standard of care.Nearly all H1N1 A/Brisbane/59/2007-like strains have been reported to beresistant to oseltamivir due to an H274 mutation within theneuraminidase gene (Centers for Disease Control and Prevention, MorbMortal, Wkly Rep 58, 115-119 (2009); incorporated by reference herein),while other strains remain susceptible to oseltamivir. Interestingly,all four influenza virus strains were either not inhibited or partiallyinhibited by oseltamivir, whereas M8 treatment of influenzavirus-infected cells with M8 at a significantly lower molarity (^(˜)20fmol) completely inhibited NS1 protein expression (FIG. 20A).Correspondingly, replication of the oseltamivir-resistant influenzavirus strain, as measured by plaque assay, indicated thatoseltamivir-treated cells were unable to reduce viral titer, whereas M8at concentrations as low as 0.1 ng/ml reduced viral titer by^(˜)2-log-fold (FIG. 20B, top right). Although the other three strainswere susceptible to oseltamivir, only cells treated with M8 showedsignificantly reduced viral titers. These results indicate that M8exhibits an enhanced broad-range antiviral activity against multipleunrelated influenza strains, including a drug-resistant strain ofinfluenza virus.

Prophylactic and Therapeutic Administration of M8 Protects Against ViralReplication:

To ensure that M8 provided long-lasting protection against infection,cells treated with M8 were infected with influenza virus or DenV for upto 72 h. At 3 days postinfection, cells were still protected from viralinfection, indicating that M8 had extended antiviral activity (FIGS. 21Ato 21C). Additionally, viral replication was significantly controlledwhen M8 was transfected therapeutically, up to 4 h postinfection (FIGS.21D and 21E), although prophylactic treatment was most effective atblocking viral replication. Cells infected with DenV had the lowestpercentage of infected cells when they were treated with M8 immediatelyfollowing a 1-h infection period, with an increase of infected cellswith each subsequent treatment (FIG. 21D). At 1 hour postinfection, thepercentage of DenV-positive cells was reduced from 33.7% to 9.3%,followed by increases in infected cells to 17.7%, 27.3%, and 32.0% at 2,4, and 8 h postinfection, respectively. In the context of influenzavirus infection, NS1 expression occurred at 1 hour postinfection with anincrease in NS1 viral protein expression as time progressed (FIG. 21E),recapitulating the results established in the DenV model. These resultsfurther characterize the protective nature of M8 as both prophylacticand therapeutic, resulting in a sustained antiviral protection andprevention of viral spread over time.

M8 5′-pppRNA Protects Mice from Lethal Influenza Virus and ChikungunyaVirus Infection:

To determine the antiviral properties of M8 in vivo, an equal amount ofWT, M5, or M8 5′-pppRNA was injected intravenously 24 h prior to and onthe day of lethal H5N1-reassorted influenza virus challenge. Untreated,infected mice succumbed to influenza infection by day 15, while at day20, at least some mice from each 5′-pppRNA treatment group survived;animals treated with M8 had the highest survival rate (80%), compared tothose mice in the WT and M5 treatment groups (20% and 40%, respectively)(FIG. 22A). Statistical significance was achieved between the nontreatedand M8 groups (log rank test for trend; P_0.0124). All groupsexperienced weight loss, with a rebound at day 11; however, animalstreated with M8 5′-pppRNA experienced a delay in the onset of weightloss and lost less weight compared to the control group (FIG. 22B).Although the M8 group showed symptoms of influenza-like illness, asdetermined by appearance and activity, the onset of symptoms was delayedand symptoms were milder compared to the other treatment groups, andmice recovered fully (FIG. 22C).

Finally, lung viral titers as determined by plaque assay revealed a2-log decrease in viral replication in M8-treated mice, a 1.5-logdecrease in M5-treated mice, and almost no difference in WT-treated mice1 day postinfection compared to controls (FIG. 22D). At 3 and 5 dayspostinfection, lung viral titers increased across all groups, but thelowest virus titers were observed in mice treated with M8.

In complementary in vivo studies, a chikungunya virus (CHIKV) infectionmodel (Pal P et al, J Virol 88, 8213-8226 (2014); incorporated byreference herein) was used to examine the antiviral effect of M8 onchikungunya-associated footpad swelling and viremia as well as surrogatemarkers for CHIKV arthritis and pathogenesis. Over 14 days, the percentchange in foot swelling, a phenotypic result of viral infection, wasreduced in mice treated with M8, while footpad swelling in untreatedmice peaked at day 7 (FIG. 22E). Analysis of viral RNA in serum revealedan ^(˜)1.5-log decrease in virus titer in M8-treated mice versus controlRNA-treated mice (FIG. 22F). These data demonstrate that prophylactictreatment with low-dose M8 5′-pppRNA protected mice from lethalinfluenza virus infection, reduced CHIKV induced arthritic swelling, andreduced viral yield of both viruses by 1 to 2 logs, compared to othertreatment groups.

Stimulation of the evolutionarily conserved RIG-I pathway usingoptimized RIG-I agonists to induce antiviral, inflammatory, and immunemodulatory gene networks is an attractive strategy for the developmentof novel antiviral compounds. Indeed, several studies have shown thatnucleic acids, such as antisense oligonucleotides and siRNA, arepromising agents for highly pathogenic RNA viruses, such as influenzavirus, Ebola virus, and West Nile virus (Spurgers K B et al, AntiviralRes, 78, 26-36 (2008); incorporated by reference herein), yet many arerestricted to targeting steps of viral replication and do not exploitthe innate immune response of the host. Furthermore, RIG-I agonists havethe advantage of dual functionality, acting as a specific sensor thattriggers the innate immune response and as a direct antiviral factorwhich can counteract the interaction of polymerase with pregenomic RNAin response to HBV infection (Sato, 2015 supra), suppressing viralreplication. The ability to inhibit viral replication of dengue,influenza, and chikungunya viruses in vitro and in vivo highlights theefficacy of M8 5′-pppRNA and potential development of an RNA-basedtherapeutic.

It was previously demonstrated that a 5′-pppRNA derived from the 5′ and3′ UTRs of the VSV genome blocked replication of a broad spectrum ofviruses and stimulated a large and unique transcriptome profile ofinflammatory and antiviral genes (27, 28). We hypothesized that arationally designed RNA agonist with enhanced antiviral activity couldbe constructed through deliberate sequence and structure alterationswhile maintaining the necessary requirements of a RIG-I ligand. In theinitial generation of RIG-I agonists, a 59-nucleotide hairpin structurewith maximal complementarity and free of loops, termed M5, was found toenhance cytokine production and inhibit viral replication compared tothe original WT when human epithelial lung cells were pretreated withthe RNA. An RNA sequence with similar structure but shorter length (M4)was the least effective of the newly modified sequences, confirming thata minimum length is required for RIG-I stimulation (Binder M et al, JBiol Chem 286, 27278-27287 (2011); Kato H et al, J Exp Med 205,1601-1610 (2008); Patel J R et al, EMBO Rep 14, 780-787 (2013); and UzriD and Gehrke L, J Virol 83, 4174-4184 (2009); all of which areincorporated by reference herein. However, further elongation of the M5sequence enhanced the inflammatory and antiviral properties 2 log-foldcompared to the WT (FIG. 16). At concentrations as low as 10 pg/ml, M8completely abrogated dengue virus and influenza virus replication, andat comparable concentrations, M8 strongly induced cytokines andantiviral genes.

In primary human dendritic cells, replication of both dengue andinfluenza viruses was inhibited when cells were pretreated with5′-pppRNA, accompanied by the induction of virtually all antiviral andinflammatory genes screened by high-throughput quantitative PCR (qPCR).Previously we showed that WT 5′-pppRNA induced a unique transcriptomeprofile compared to IFN-α-2b treatment, including the upregulation ofCCL3, CCL5, IL-29, CXCL10, and IL-6 (Goulet et al, 2013 supra). M8induced a striking antiviral response compared to WT treatment (FIGS.19A and 19B), suggesting that M8 functions to broadly activate theinnate immune response even in the absence of viral infection. Incontrast to the previously published study (Goulet et al, 2013 supra),here we used considerably lower concentrations of M8 to induce a morerobust inflammatory response compared to the original WT 5′-pppRNA.Induction of an antiviral response was increasingly heightened betweenthe 59-nucleotide M5 and the 99-nucleotide M8, again highlighting theimportance of RNA length to activate RIG-I. Additionally, the immenseupregulation of the expression of nearly all type I and III interferons,inflammatory cytokines and chemokines, and interferon-stimulated genestested suggests that M8 induces a complete interferon response inpreparation of viral infection. Additionally, this robust triggering ofthe innate immune response resulted in a concomitant antiviralprotection in A549 cells (FIGS. 16A-16G) and Mo-DCs (FIGS. 19A-19F). Theenhanced antiviral properties of M8 in vitro were confirmed in vivo, asM8 administration increased survival of mice challenged with a lethaldose of influenza virus and decreased viral titers in influenza virus-and chikungunya virus-infected mice (FIGS. 22A-22F).

Prophylactic administration of M8 not only enhanced viral inhibition invitro and in vivo compared to the prototype VSV derived WT 5′-pppRNA butalso surpassed the antiviral activity of oseltamivir, the currentstandard of care for influenza. Due to concerns about widespreadresistance of circulating influenza viruses to current antiviral drugssuch as zanamivir and oseltamivir, the constant need to monitorinfluenza virus strains for these changes in resistance and, moreimportantly, to develop novel, more effective treatments without sideeffects remains. M8 reduced viral protein expression and titers of aknown oseltamivir-resistant strain, H1N1 A/Brisbane/59/2007, at a lowconcentration (FIGS. 20A and 20B), indicating that it would be aneffective therapeutic to control viral infection of drug-resistantstrains.

One of the more interesting conclusions of these studies is the apparentrole that sequence plays in enhancing antiviral activity. Others haveshown differences in activity with regard to RNA origin, motifs, length,and structure, whereas the effect of primary RNA sequence has not beenconsidered. Primary sequence modification was sufficient todifferentially trigger an innate response, although increased length ofthe sequence via additional UA pairings also enhanced the response. Ithas been reported that a poly(U) motif is required to produce aninterferon response that establishes an antiviral state (Hwang S Y etal, Nucleic Acids Res 40, 2724-2733 (2012); incorporated by referenceherein), and others observed enhanced activity with a poly(U) sequence,although they determined that it was not necessary to elicit a response(Uzri and Gehrke 2009 supra; Saito T et al, Nature 454, 523-527 (2008);and Schnell G et al, PLoS Pathog 8, e1002839 (2012); both of which areincorporated by reference herein). However, RIG-I-mediated immunity isnot dependent on this specific moiety, as a number of RNA moleculestrigger RIG-I without it. Here we reveal that RNA disruption of any partof the poly(U) sequence reduced antiviral and inflammatory activity,suggesting a crucial role for the poly(U) moiety in RNA-RIG-I binding(FIG. 17A-17E).

The results described in this study demonstrate that 5′-pppRNA can berationally designed to achieve a maximal RIG-I mediated protectiveantiviral response against a number of RNA viruses, including influenzastrains not inhibited by oseltamivir, in vitro. In vivo studiesdemonstrate that novel RIG-I agonists reduced viral load and improvedsurvival of animals when they were treated with the 5′-pppRNA and thensubsequently infected with influenza virus or chikungunya virus. Thecurrent global need for antiviral treatment persists, as there ispresently no cure for dengue or chikungunya virus infections and currenttreatments for influenza are often ineffective and can produce harmfulside effects.

Additionally, the compositions described herein can be used as a vaccineadjuvant Ichinohe T et al, J Virol 79, 2910-2919 (2005); Kulkarni R R etal, J Virol 88, 13990-14001 (2014); and Martinez-Gil L et al, J Virol87, 1290-1300); all of which are incorporated by reference herein) andcould fill the growing need for novel therapeutics against infectiousdiseases.

Materials and Methods:

In vitro transcription and gel analysis. RIG-I agonists were synthesizedby designing complementary forward (F) and reverse (R) primers with a T7promoter (Integrated DNA Technologies). Primers are exemplified by SEQID NOs: 18-43. Primers were annealed then synthesized with an in vitrotranscription kit (Ambion) for 16 h. RNA transcripts were DNase digestedfor 15 min at 37° C. and then purified with an miRNeasy kit (Qiagen).RNA was analyzed on a denaturing 15% Tris-borate-EDTA (TBE)-ureapolyacrylamide gel (Bio-Rad) following digestion with 50 ng/μl of RNaseA (Ambion) or 100 mU/μl of DNase I (Ambion) for 30 min. Controlwild-type RNA is the dephosphorylated form of the WT sequence purchasedfrom IDT. Secondary structure was predicted using the RNAfold Webserver(University of Vienna).

Cell Culture, Transfections, and Luciferase Assays:

Lung epithelial A549 cells were grown in F12K (ATCC) supplemented with10% fetal bovine serum (FBS) (Access Cell Culture). Transfection of RNAand small interfering RNA (siRNA) in A549 cells was performed withLipofectamine RNAiMax® (Invitrogen) for 18 to 24 h and 48 h,respectively. Transfections of RNA and siRNA in monocyte-deriveddendritic cells (Mo-DCs) were performed with HiPerFect transfectionreagent (Qiagen). Poly(I⋅C) LMW was purchased from Invivogen. For siRNAknockdown, A549 cells were transfected with 30 pmol of control siRNA(sc-37007), human RIG-I (sc-61480), TLR3 (sc-36685), MDA5 (sc-61010),TLR7 (sc-40266), or TLR8 (sc-40268) (Santa Cruz Biotechnologies) usingLipofectamine RNAiMax according to the manufacturer's guidelines. Forluciferase assays, 200 ng IFN-γ/pGL3 and 100 ng pRL-TK plasmids werecotransfected with 5′-pppRNA using Lipofectamine RNAiMax for 24 h.Reporter gene activity was measured by a dual-luciferase reporter assay(Promega) according to the manufacturer's instructions. Relativeluciferase activity was measured as fold induction. Oseltamivir waspurchased from AvaChem Scientific.

Monocyte Isolation and Differentiation into Monocyte-Derived DendriticCells:

Human peripheral blood mononuclear cells (PBMC) were isolated from buffycoats of healthy, seronegative volunteers in a study approved by theinstitutional review board (IRB) and by the Vaccine & Gene TherapyInstitute of Florida (VGTI-FL) Institutional Biosafety Committee(2011-6-JH1). Written informed consent approved by the VGTI-FL, Inc.,ethics review board (FWA number 161) was provided to study participants.Research conformed to ethical guidelines established by the ethicscommittee of the Oregon Health and Science University (OHSU), VGTI, andMartin Health System. Briefly, PBMC were isolated from freshly collectedblood using Ficoll-Paque Plus medium (GE Healthcare Bio) as per themanufacturer's instructions. CD14 monocytes were isolated by positiveselection using CD14 microbeads and a magnetic cell separator as per kitinstructions (Miltenyi Biotech). Purified CD14 monocytes were culturedfor 7 days in 100-mm dishes (15×10⁶ cells) in 10 ml of complete monocytedifferentiation medium (Miltenyi Biotech). On day 3, the medium wasreplenished with fresh medium. Differentiation was confirmed by flowcytometry analysis on day 7. CD14^(low) CD1a^(high) DC-SIGN^(high) cellswere used for experiments.

Quantitative Real-Time RT-PCR:

Total RNA was isolated from cells using an RNeasy kit (Qiagen) accordingto the manufacturer's instructions. RNA was reverse transcribed usingthe SuperScript VILO cDNA synthesis kit (Invitrogen) according to themanufacturer's instructions. PCR primers were designed using Roche'sUniversal Probe Library Assay Design Center and purchased fromIntegrated DNA Technology. Quantitative RT-PCR was performed on aLightCycler 480 Probes Master (Roche.) All data are presented as arelative quantification with efficiency correction based on the relativeexpression of target gene versus GAPDH as the invariant control.

Fluidigm BioMark Assay:

The 5′-pppRNA BioMark experiment was performed with Mo-DCs derived from3 independent healthy donors. Total RNA and cDNA were prepared asdescribed above. cDNA along with the entire pool of primers werepreamplified for 18 cycles using TaqMan PreAmp master mix as per themanufacturer's protocol (Applied Biosystems). Preamped cDNA was treatedwith Exonuclease I (New England BioLabs) and then combined with 2×FastStart TaqMan Probe MasterRoche), GE sample loading buffer(Fluidigm), and Taq polymerase (Invitrogen). Assays were prepared with 2assay loading reagent (Fluidigm), primers (IDT), and probes (Roche).Samples and assays were loaded in their appropriate inlets on a 48.48BioMark chip. The chip was run on the BioMark HD System (Fluidigm) for40 cycles. Raw cycle threshold (C_(T)) values were calculated by thereal-time PCR analysis software (Fluidigm), and software-designatedfailed reactions were discarded from analysis. All data are presented asrelative quantifications with efficiency corrections based on therelative expression of target gene versus the geomean of(GAPDH+actin+β2-microglobulin) as the invariant control. The n-folddifferential expression of mRNA gene samples was expressed as 2^(−ΔΔCT).The heatmaps were produced with the pheatmap Pretty Heatmaps package andR package version 0.7.7 (http://CRAN.R-project.org/package_pheatmap).Gene level expression is shown as 2^(−ΔΔCT) or genewise standardizedexpression (Z score). The sequences of forward (F) and reverse (R)primers used are SEQ ID NOs: 45-111.

Immunoblot Analyses:

Whole-cell extracts were separated in 4 to 20% acrylamide Mini-ProteanTGX precast gels (Bio-Rad) by SDS-PAGE and transferred to anImmobilon-PSQ polyvinylidene difluoride (PVDF) membrane (Millipore) for1 h at 100 V in a buffer containing 30 mM Tris, 200 mM glycine, and 20%methanol. Membranes were blocked for 1 h at room temperature in blockingbuffer (Odyssey) and then probed with the following primary antibodies:anti-RIG-I (EMD Millipore), anti-IFIT1 (Thermo Fisher Scientific),anti-ISG56 (Cell Signaling), anti-STAT1 (Cell Signaling), anti-pIRF3S396 (Cell Signaling), anti-IRF3 (Cell Signaling), anti-TLR3 (CellSignaling), anti-MDA5 (Cell Signaling), anti-β-actin (Odyssey),anti-dengue virus (DenV) 2E protein (Santa Cruz Biotechnology), andanti-NS1 (Santa Cruz Biotechnology). Antibody signals were detected byimmunofluorescence using the IRDye 800CW and IRDye 680RD secondaryantibodies (Odyssey) and the LI-COR imager (Odyssey).

Flow Cytometry Analysis:

The percentage of dengue virus-infected cells was determined by standardintracellular staining (ICS) using a mouse IgG2a monoclonal antibody(MAb) specific for dengue virus E protein (clone 4G2) followed bystaining with a secondary anti-mouse antibody coupled to phycoerythrin(PE) (BioLegend). pSTAT1 geomean fluorescence was determined by PhosFlowstaining as previously reported (Olagnier D et al, PLoS Pathog 10:e1004566 (2014); incorporated by reference herein) with pSTAT1 Y701Pacific Blue antibody (BD Biosciences). Cells were analyzed on an LSRIIflow cytometer (Becton Dickinson). Calculations and population analyseswere done using FACSDiva software.

Virus Production and In Vitro Infection:

Dengue serotype 2 strain New Guinea C (NGC) was used to infect confluentmonolayers of C6/36 insect cells at a multiplicity of infection (MOI) of0.5. Virus was allowed to adsorb for 1 h at 28° C. in serum-freeDulbecco's modified Eagle medium (DMEM). Serum-free DMEM was used towash the monolayer and then replaced with DMEM-2% FBS. After 7 days ofinfection, the medium was harvested and cleared by centrifugation(1,100×g for 10 min), and the supernatant was concentrated bycentrifugation (1,100 g) through a 15-ml Amicon centrifugal filter unit(Millipore). The virus was concentrated by ultracentrifugation on asucrose density gradient (20% sucrose cushion) using a SorvallWX100Ultracentrifuge (ThermoScientific) for 2 hours at 134,000 g and 10° C.with the brake turned off. Concentrated virus was then washed to removesucrose using a 15-ml Amicon tube. After 2 washes, the virus wasresuspended in DMEM-0.1% bovine serum albumin (BSA). Titers of denguestocks were determined by fluorescence activated cell sorting (FACS)after infection of Vero cells and immunofluorescence staining ofintracellular dengue E protein 24 h postinfection. For dengue viruschallenge experiments, A549 cells and Mo-DCs were infected using denguevirus at an MOI of 0.5 in serum-free medium for 1 h at 37° C. Medium wasreplaced with complete medium for 24 h prior to analysis.

For in vitro influenza virus challenge experiments, A549 cells andMo-DCs were infected with various influenza virus strains (MOI of 0.2 or2) in a small volume of serum-free medium for 1 h at 37° C. Medium wasreplaced with complete medium for 24 h prior to analysis.

Reassortant H5N1 influenza virus (H5N1-PR8) was generated usinghemagglutinin (HA) and neuraminidase (NA) genes derived from the H5N1virus [HA from influenza A/Vietnam/1203/2004 and NA from influenzaA/Thailand/1(KAN-1)/2004]. The internal viral proteins were derived fromthe A/Puerto Rico/8/1934 (PR8) mouse-adapted influenza A virus. TheH5N1-PR8 reassortant viruses were propagated using MDCK cells. Allprocedures with live dengue and influenza viruses were performed in abiosafety level 2 facility at the Vaccine & Gene Therapy Institute ofFlorida.

Plaque Assay:

The plaque assay was performed on supernatants from influenza-infectedA549 cells. MDCK cells in 6-well plates were grown to confluence andwashed twice with DMEM containing 1% penicillin streptomycin (LifeTechnologies). Serial dilutions of virus (1:10) were inoculated on MDCKcells in a volume of 100 μl and adsorbed for 1 h at room temperature,with rocking every 15 min. Wells were washed twice with DMEM containingantibiotics. Leibovitz's L-15 medium (2, with L-glutamine) (Cambrex) wasprepared with HEPES, 7.5% sodium bicarbonate, gentamicin, and TPCK(tosylsulfonyl phenylalanyl chloromethyl ketone)-trypsin (0.6 mg/ml)(Sigma-Aldrich), combined with 1.6% agarose, and overlaid on infectedcells. Plates were incubated at 37° C. and monitored daily for plaques.At 48 h postinfection, plaques were stained with 1% crystal violet-20%ethanol and counted, and viral titers were calculated.

In Vivo Administration of 5′-pppRNA and Viral Infection:

BALB/c mice (6 to 8 weeks of age; Jackson Laboratories) were housed incage units, fed ad libitum, and cared for under USDA guidelines forlaboratory animals. Prior to 5′-pppRNA injections and viral challenge,mice were anesthetized with IsoSol (Patterson Veterinary), and 5 μg ofpurified 5′-pppRNA was injected intravenously via a tail vein. The5′-pppRNA was complexed with in vivo-jetPEI (PolyPlus, France) at an N/Pratio of 8 according to the manufacturer's instructions. Mice were thenchallenged with H5N1-RE (5,000 PFU in 50 μl of phosphate-buffered saline[PBS]). Animals were monitored for survival and morbidity (weight loss,ruffling fur, hunched back, and lethargy) each day during the viralchallenge. Lungs were isolated from mice postmortem and snap-frozen indry ice/ethanol bath. DMEM (10 volumes to grams) of was then added tothe tissue placed in a 0.7-μm cell strainer in a petri dish, and thesample was muddled until it had been broken down. The remaining liquidwas collected from the petri dish in a sample tube for stock lunghomogenate sampling. All procedures with reassortant influenza virusH5N1-PR8 were performed in a biosafety level 2 facility at the Vaccine &Gene Therapy Institute of Florida.

Control RNA and M8 5′-pppRNA were administered to adult mice using aprotocol similar to that of the influenza virus infection in vivo model.Mice were injected intraperitoneally with 2 μg RNA in combination within vivo JetPEI for 24 h and then infected with 1,000 PFU chikungunyavirus via footpad injection in 20 μl RPMI. Treatments were assessed viacaliper for footpad swelling or viral titer of blood as determined byplaque assays on confluent monolayers of Vero cells as previouslyreported (Pal et al, 2014 supra). All procedures with chikungunya viruswere performed at Oregon Health and Science University.

Statistical Analyses:

Values were expressed as the means±standard errors of the means, andstatistical analysis was performed using Prism software (GraphPadsoftware) or Microsoft Excel using an unpaired, two tailed Student's ttest to determine significance. Differences were considered significantat a P value of <0.05.

Example 4—Enhanced Influenza VLP Vaccination with a StructurallyOptimized RIG-I Agonist as Adjuvant

The molecular interaction between viral RNA and the cytosolic sensorRIG-I represents the initial trigger in the development of an effectiveimmune response against infection with RNA viruses, resulting in theactivation of innate antiviral factors and subsequent induction ofadaptive responses. In the present study, the adjuvant properties of asequence-optimized 5′pppRNA RIG-I agonist (termed M8 herein) wereexamined in combination with influenza virus-like particles (VLP) asimmunogens. A formulation comprising both VLP and M8 resulted in ahigher antibody response than that of VLP alone. Furthermore, thisformulation protected mice against lethal reassortant H5N1 influenzachallenge more readily than controls. M8-VLP immunization also resultedin long term protective responses against influenza infection in mice.M8-VLP immunization also increased endpoint and antibody titers andinhibited influenza replication in lungs, when compared with otheradjuvants such as Alum, AddaVax, and poly(I:C).

Immunization with M8-VLP stimulated a T_(H)1-biased CD4 T cell response,as determined by higher T_(H)1 cytokine levels in CD4 T cells andincreased IgG2 levels in sera than controls lacking M8. Collectively,these data demonstrate that RIG-I agonists are potent adjuvants. Thedevelopment of novel adjuvants to increase vaccine immunogenicity is animportant goal that seeks to improve vaccine efficacy and thus preventinfections that endanger human health and safety. This exampledemonstrates the adjuvant properties of a RIG-I agonist using influenzavirus-like particles (VLP) as antigens.

Annual vaccination with the trivalent inactivated influenza vaccine(TIV), quadrivalent inactivated influenza vaccine (QIV), or the liveattenuated influenza vaccine (LAIV) are the primary strategies forreducing the morbidity and mortality associated with human influenzainfection (Hannoun C, Exp Rev Vaccines 12, 1085-1094 (2013 and McKeageK, Drugs 73, 1587-1594 (2013); both of which are incorporated byreference herein). The protection provided by the TIV vaccine is strongin young adults, but its efficacy decreases in the elderly due toimmunosenescence, which is characterized by decreases in effector cellnumber and function, as well as alterations in production ofinflammatory and antiviral cytokines (Metcalf T U et al, Aging Cell 14,421-432 (2015); Baldwin S L et al, J Immunol 188, 2189-2197 (2012); andAtmar R L and Keitel W A, Curr Top Microbiol Immunol 333, 323-344(2009); all of which are incorporated by reference herein).Additionally, individual immune responses vary dramatically because ofmultiple factors, including the immunogen, route of administration, ageof the subject, and virus type. Thus, an additional immune stimulationis frequently necessary to enhance vaccine efficacy. With increasingemphasis on subunit and/or peptide-based immunization and the ultimateneed to develop a universal influenza vaccine, new approaches to improvevaccine efficacy are warranted (McLean H Q et al, J Infect Disdoi:10.1093/infdis/jiu647 (2014); incorporated by reference herein).

Virus-like particles (VLP) are an attractive alternative to moretraditional live-attenuated or split vaccines. VLP mimic the virus instructure and morphology, but are non-infectious, and thus possess ahigh safety profile that enhances their potential for future vaccinedevelopment against highly pathogenic strains (Schneider-Ohrum K andRoss T M, Curr Top Microbiol Immunol 354, 53-73 (2012); incorporated byreference herein). As with live, attenuated virus vaccination, VLPstimulate the immune system, leading to both humoral and cellular immuneresponses. An effective VLP-based vaccine typically includes a strongimmunogen (e.g. a viral surface glycoprotein such as hemagglutinin) anda potent adjuvant for inducing antiviral signals (Alving C R et al, CurrOpin Immunol 24, 310-315 (2012) and Osterholm M T et al, Lancet InfectDis 12, 36-44 (2012); both of which are incorporated by referenceherein). Importantly, VLP can be genetically engineered to expressvaccine antigens that represent a population of sequences and elicitcross-protective immune responses against multiple pathogens (Giles B Mand Ross T M, Exp Rev Vaccines 11, 267-269 (2012); incorporated byreference herein). Influenza VLP can be formed following co-expressionof just three viral proteins—matrix, haemagglutinin (HA), andneuraminidase (NA)—in a mammalian expression system; VLP express themajor surface influenza proteins in the same conformation as found inthe influenza virion and have been shown to stimulate a potent immuneresponse (Schneider-Ohrum and Ross, 2012 supra).

Addition of an adjuvant is a key strategy that: 1) enhancesimmunogenicity of the antigen; 2) permits a reduction in the amount ofviral epitope per vaccine (termed “antigen sparing”); and 3) stimulatesimmune responsiveness in the elderly, thus increasing vaccine efficacyin this population (Leroux-Roels I et al, Lancet 370, 580-589 (2007);incorporated by reference herein). The most commonly used FDA-approvedadjuvant is aluminum salts (Alum) although it is not included in any ofthe current influenza formulations in the US. In addition to Alum,vaccines can be formulated with adjuvants such as MF59, AF03 and AS03,vaccine antigen delivery vehicles, virosomes, (Schwendener R A, Ther AdvVaccines 2, 159-182 (2014); incorporated by reference herein), and theadjuvant combination AS04 (Lee Y N et al, Vaccine 32, 4578-4585 (2014);incorporated by reference herein) All of these adjuvants act by creatingan antigen depot, activating antigen-presenting cells, and triggering aninnate immune response by stimulation of danger signals (Reed S G et al,Nat Med 19, 1597-1608 (2013); incorporated by reference herein). Giventhe wide range of vaccine strategies currently under study, a highpriority for the development of influenza vaccines is the identificationof novel adjuvants that elicit a broad and robust immune response toincrease immunogenicity of antigens and to enhance the antigen sparingeffect (Reed, 2013 supra, Coffman R L et al, Immunity 33, 492-503(2010); and Jiang F et al, Nature 479, 423-427 (2011); both of which areincorporated by reference herein).

Influenza infection is sensed by RIG-I, a cytosolic sensor that detectsviral RNA during replication through its helicase domain (Jiang F et al,Nature 479, 423-427 (2011); incorporated by reference herein). RIG-Ialso possesses an effector caspase activation and recruitment domain(CARD) that forms a complex with the mitochondrial adaptor MAVS (KomuroA et al, Cytokine 43, 350-358 (2008); incorporated by reference herein).MAVS serves as a signaling platform for protein complexes that activatestranscription factors NF-κB, IRF3 and IRF7 Belgnaoui S M et al, CurrOpin Immuno/23, 564-572 (2011) and Loo Y M and Gale Jr. M, Immunity 34,680-692 (2011); both of which are incorporated by reference herein) thatinduce the expression and production of type I interferon (IFN), as wellas pro-inflammatory cytokines and antiviral proteins (Takeuchi O andAkira S, Cell 140, 805-820 (2010); incorporated by reference herein). Asecondary response involving hundreds of IFN stimulated genes (ISGs) isinduced by secreted IFN binding to the type I IFN receptors on adjacentcells and tissues, leading to amplification of the antiviral immuneresponse via the JAK-STAT pathway (Schoggins J W et al, Nature 472,481-485 (2011); incorporated by reference herein). This stimulation ofthe innate antiviral response also contributes to the maturation ofdendritic cells and clonal expansion of CD4+ and CD8+ T-cells (Longhi MP et al, J Exp Med 206, 1589-1602 (2009); incorporated by referenceherein), all of which contribute to the establishment of long-termadaptive immunity against infection. The use of natural or synthetic5′-triphosphate-containing RNA (5′pppRNA) that activate innate immunityvia RIG-I could therefore be an attractive strategy for the developmentof broad-spectrum antivirals and vaccine adjuvants.

It has been previously demonstrated that intravenous injection of ashort, double-stranded RNA containing a 5′triphosphate (5′ppp) moiety,derived from the 5′ and 3′-untranslated regions of the vesicularstomatitis virus VSV genome (WT 5′pppRNA) stimulated innate responses inlungs and protected mice from lethal H1N1 influenza challenge (Goulet ML et al, PLoS Pathol 9, e1003298 (2013); incorporated by referenceherein). Transcriptional profiles of lung epithelial cells treated invitro with the oligoribonucleotide termed WT 5′pppRNA herein identifiedoverlapping and unique transcriptional signatures associated with genescapable of mobilizing multiple arms of innate and adaptive responses.Specific modifications in the structure of theoligoribonucleotide—modifications to the primary RNA sequence thateliminated mismatch in the double stranded RNA region—removed thepanhandle structure and introduced additional bases—resulted in a59-nucleotide double-stranded RNA structure (M5) with improved antiviralproperties compared to WT 5′pppRNA. Further improvement of M5 antiviralproperties was achieved by the addition of AU base pairs to increase thelength of the dsRNA stem structure. The resulting sequence-optimizedagonist, M8, further increased breath and magnitude of the antiviral andinflammatory response observed in primary human dendritic cells comparedto WT 5′pppRNA or M5. As an antiviral agent, M8 efficiently inhibitedinfluenza and dengue virus replication in vitro and decreased bothchikungunya and influenza virus replication in vivo.

Because of its capacity to stimulate a potent antiviral and inflammatoryresponse, we hypothesized that M8 may also function as an adjuvant in aVLP-based influenza vaccine formulation. In the present study, wedemonstrate that M8-VLP increased antibody levels, protected againstlethal influenza H5N1 challenge, stimulated formation of germinal centerB cells, and induced a T_(H)1-predominant cellular immune phenotype uponvaccination. These results illustrate that agonist-specific stimulationof the RIG-I pathway can be used as an adjuvant in influenza VLPvaccination and dramatically improve humoral and cellular mediatedprotective responses against influenza challenge.

In Vitro Synthesis of 5′pppRNA.

RIG-I agonists were synthesized using in vitro RNA transcription kit(Ambion) as previously described (Goulet et al, 2013 supra). RNAtranscripts were digested with DNase for 15 minutes at 37° C. thenpurified using miRNeasy kit (Qiagen). Integrity of the purified 5′pppRNAwas analyzed on a denaturing 15% TBE-urea polyacrylamide gel (Bio-Rad)and compared to the 5′pppRNA digested with RNase A or DNase (Ambion).

Isolation and Transfection of Monocyte-Derived Dendritic Cells (MDDC).

Human peripheral blood mononuclear cells (PBMC) were isolated from buffycoats of healthy volunteers. PBMC were isolated using the Ficoll-Paque™PLUS medium (GE Healthcare Bio). CD14+ monocytes were isolated bypositive selection using CD14 microbeads (Miltenyi Biotech) and amagnetic cells separator. Purified CD14+ monocytes were cultured for 7days in in 10 mL of complete monocyte differentiation medium (MiltenyiBiotech). On day 3, the medium was replenished with fresh medium. Onlycells with the CD14^(lo)CD1a^(hi)DC-SIGN^(hi) phenotype after 5-7 daysdifferentiation were used in subsequent experiments. MDDCs weretransfected with various amounts of WT, M5, or M8 5′pppRNA or poly (I:C)for 24 h using HiPerfect Transfection Reagent (Qiagen) for 24 h. MDDCswere stained for 5 minutes with human TruStain FcX (Biolegend) followedby staining with CD83-PE (Biolegend), CD86-Pacific Blue (Biolegend),CD80-PE, or CD40-PE, for 15 minutes at 4° C. Cells were analyzed on anLSRII flow cytometer (Becton Dickinson). Calculations and populationanalyses were done using FACS Diva software.

Quantitative Real-Time RT-PCR.

Total RNA was isolated from cells using RNeasy kit (Qiagen) and RNA wasreverse transcribed using the SuperScript® VILO cDNA synthesis kit(Invitrogen). PCR primers were designed using Roche's Universal ProbeLibrary Assay Design Center (www.universalprobelibrary.com).Quantitative RT-PCR was performed on a LightCycler® 480 Probes Master(Roche.) All data are presented as a relative quantification withefficiency correction based on the relative expression of target geneversus GAPDH as the invariant control. The sequences of forward andreverse primers used are listed as SEQ ID NOs: 112-127 above.

Virus Propagation and Challenge:

Influenza reassortant mouse adapted H5N1 virus (H5N1) expressing H5haemagglutinin (HA) A/Vietnam/1203/2004 and neuraminidase (NA)A/Thailand/1(KAN-1)/2004) and internal viral genes from mouse adaptedA/Puerto Rico/8/1934 (PR8), was propagated using MDCK cells aspreviously described (Bright R A et al, PLoS One 3, e1501 (2008);incorporated by reference herein). In animal challenge experiments,anesthetized female BALB/c mice were infected intranasally with thelethal dose (5×10³ PFU in 50 μL PBS) of the H5N1. This dose representsapproximately 50 LD₅₀ doses in mice.

Virus Like Particle Vaccine:

H5N1 virus like particles were purified from HEK293T cells which weretransfected using Lipofectamine2000 (Invitrogen) with 5 μg of eachplasmid DNA expressing H5N1 A/Vietnam/1203/2004 HA and H5N1A/Thailand/1(KAN-1)/2004 NA (codon optimized); and 10 μg of plasmid DNAexpressing HIV gag. Cells were incubated for 72 h at 37° C. andsupernatants containing VLP were collected, sterile filtered, andpurified by centrifugation at 100,000×g through a 20% glycerol cushionand resuspended in PBS. Total protein was quantified using BCA proteinassay (Thermo Fisher Scientific) and VLP were aliquoted in PBS andstored at −80° C. HA content was quantified by densitometry as describedpreviously (Giles B M and Ross T M, Vaccine 29, 3043-3054 (2011);incorporated by reference herein).

Immunization:

BALB/c mice (6-8 weeks of age, Jackson Laboratories) were housed in cageunits, fed ad libitum, and cared for under USDA guidelines forlaboratory animals. For immunization, mice were anesthetized with IsoSol(Patterson Veterinary) and immunized via the intramuscular route (IM)with 0.5 μg-2 μg (based on HA content) of purified VLP (in 50 μl PBS)with or without 0.1 μg-5 μg 5′pppRNA as adjuvant and then challenged atweek 3. The 5′pppRNA was delivered with in vivo-jetPEI (PolyPlus,France) at an N/P ratio of 8 according to the manufacturer instructions.Imject Alum (Fishersci, Pittsburgh, Pa.) and AddaVax (Invivogen, SanDiego, Calif.) were added to 50% volume of VLP in PBS solution permanufacturer's recommendation. Animals were monitored for survival andmorbidity weekly during the immunization regimen and each day during theviral challenge. Blood samples for serological analysis were collectedfrom anesthetized mice via retro-orbital sinus. Blood was allowed toclot at room temperature and sera was removed and frozen at −80° C.after centrifugation.

Sickness Score.

After infection, mice were monitored daily for weight loss, diseasesigns and death for up to 21 days after infection. Individual bodyweights, sickness scores and death were recorded for each mouse afterinoculation. The sickness score was generated by evaluating activity(0=normal, 1=reduced, 2=severely reduced), hunched back (0=absent,1=present) and ruffled fur (0=absent, 1=present) as described previously(27). The final score was the addition of each individual scoreresulting in the minimum score 0 for a healthy mouse and 1-4 for a sickmouse.

Virus Plaque Assay.

Lungs were isolated from mice post-mortem and snap frozen in dryice/ethanol bath. Dulbecco's modified Eagle medium (DMEM, 10 volumes tograms) of was then added to the tissue placed in a 0.7 μm cell strainerin a petri dish and sample was muddled until tissue was fullydisaggregated. Remaining liquid was collected from petri dish in sampletube for stock lung homogenate sample. Confluent MDCK cells plated in6-well tissue culture plates were inoculated with 0.2 ml of virus orlung cell supernatant serially diluted (1:10) in DMEM. Virus wasadsorbed for 1 h, with shaking every 15 min. Wells were overlaid with1.6% (w/v) Bacto agar (BD Diagnostic Systems) mixed 1:1 with L-15 media(Cambrex) containing 1% Pen Strep (Life Technologies), with 0.6 mg/mlTPCK-Trypsin (SigmaAldrich). Plates were incubated for 2 days at 37° C.and plaques were visualized with crystal violet.

Hemagglutination Inhibition Activity.

The hemagglutination inhibition (HAI) assay was used to assessfunctional antibodies to the HA able to inhibit agglutination of horsered blood cells. The procedure was adapted from the Center for DiseaseControl influenza surveillance manual and performed as previouslydescribed (Bright et al 2008 supra).

Serological Assays.

ELISA plates (BD biosciences) were coated with 25 ng per well ofpurified recombinant hemagglutinin encoded by A/Vietnam/1203/2004 incarbonate buffer pH 9.4 containing 5 μg/mL BSA Fraction V at 4° C.overnight. Plates were then blocked with ELISA blocking buffer (PBScontaining 5% BSA Fraction V, 2% bovine gelatin and 0.05% Tween 20)for >90 min at 37° C. Serial dilutions of immune sera were incubatedfor >90 min and plates washed five times with PBS. Total HA-specific IgGwas detected using horseradish peroxidase conjugated Goat anti-mouse IgG(γ-chain specific) (Southern Biotech, Birmingham, Ala.) diluted to1:2500 in ELISA blocking buffer. Following additional PBS washes,2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) substratewas added and plates incubated 10-20 min at 37° C. Colorimetricconversion was measured as the optical density (O.D. at 414 nm) by aspectrophotometer (BioTek, Winooski, Vt., USA). O.D. values from BSAonly coated wells were subtracted to determine HA-specific reactivity.HA-specific IgG subclasses were measured in sera samples diluted to 1:50in ELISA blocking buffer and detected using horseradish peroxidaseconjugated goat anti-mouse IgG1, IgG2a IgG2b, and IgG3 antibodies(Southern Biotech, Birmingham, Ala.) diluted to 1:1000 in blockingbuffer.

Assessment of T and B Cell Responses:

Spleens from mice immunized intraperitoneally 8 days prior with 1 μg VLP(based on HA content) were harvested in cold RPMI. Spleens weremechanical dissociated through 70 μm cell strainers and the resultingsingle-cell suspension washed in cold RPMI. The cell suspensions werethen incubated with ACK buffer to lyse RBC and counted following awashing step. For assessment of B cell responses, 2-5×10⁶ splenocyteswere stained directly ex vivo. For assessment of T cell responses, 5×10⁶splenocytes were cultured in RPMI containing vehicle, VLP (1 μg/mL) orthe HA-derived peptide (IYSTVASSL) (10 μg/mL) for 21 h. Brefeldin A(Ebioscience) was added 6 hr prior to harvest. The following monoclonalantibodies were used: FITC anti-mouse TNF-α (MP6-XT22) PE anti-mouse11-2 (clone JES6-5H4), PE-Cy7 anti-mouse IFN-γ (clone XMG1.2), PacificBlue anti-mouse CD8a (clone 53-6.7), APC anti-mouse CD4 (clone GK1.5),PE-Dazzle594 anti-mouse B220 (clone RA3-6B2), Pacific Blue anti-mouseCD44 (clone IM7), PerCP anti-mouse CD19 (clone eBio1D3), FITC anti-mouseCD38 (clone 90), and Alexa Fluor647 anti-mouse IgG1 (clone RMG1-1,1:500) (all from BioLegend). Additional antibodies were biotinanti-mouse CD95 (clone Jo2) and PE anti-CD138 (clone 281-2) (both fromBD biosciences). Both LIVE/DEAD fixable dead cell stain (Invitrogen) andintracellular staining using BD Cytofix/Cytoperm™ Kit were performedaccording to the manufacturers' instructions.

Histology.

Lungs were harvested and prepared for immunohistochemistry using amodified protocol (Olagnier D and Hiscott J, Hepatology 59, 1225-1228(2014); incorporated by reference herein). After euthanasia, the chestcavity was opened and the lungs were gently inflated intratracheallywith 4° C. 4% paraformaldehyde in PBS, removed and immersed in 4%paraformaldehyde at 4° C. overnight. The next day, the solution wasreplaced with 70% ethanol and tissues were kept at 4° C. for up to 2weeks. Tissue sections were embedded in paraffin, sectioned into slices(^(˜)5 μm in thickness), and stained with hematoxylin and eosin (H&E) orleft unstained. Tissue sections were imaged with a QimagingMicropublisher 5.0 RTV digital camera on an Olympus BX61 fluorescencemicroscope. To quantify the number of apoptotic lung cells,representative sections were deparaffinized and rehydrated in xylene andgraded alcohols, respectively, using standard procedures, and TUNELassay was performed according to manufacturer's instructions (Roche,Mannheim, Germany). The percentages of TUNEL-positive cells within thetissue sections were determined by counting at least 100 cells each fromeight randomly selected fields.

Statistical Analyses:

Values are expressed as mean±SEM, and statistical analysis was performedusing PRISM software (GraphPad software) using one-way ANOVA followed byTukey post-hoc test to determine significance.

M8 Potentiates DC Maturation and Anti-Viral Signaling in Human MDDC.

The most efficient antigen-presenting cells are mature, immunologicallycompetent dendritic cells (DC) (Schraml B U and Reis E S C, Curr OpinImmunol 32C, 13-20 (2014); incorporated by reference herein) and it isnow accepted that the adjuvant component of vaccines contributes tovaccine efficacy by triggering DC maturation and antigen presentation(Palucka K and Banchereau J, Nat Rev Cancer 12, 265-277 (2012);incorporated by reference herein. To determine whether selected 5′pppRNAsequences differ in their ability to induce DC maturation ex vivo, themRNA expression of selected genes involved in DC maturation andactivation was assessed. Chemokine CCL4, activation and/orco-stimulation markers CD40, CD80, CD83 and CD86, 4-1BB, HLA-DRA,HLA-DQA, and CD74 expression were all upregulated by treatment ofprimary DC cultures with M8 (FIG. 23A). These data were confirmed bymeasuring the protein expression of several DC activation/maturationmarkers in MDDC treated with WT 5′pppRNA, M5, M8, and poly(I:C) (FIG.23A) (Yu M and Levine S J, Cytokine Growth Factor Rev 22, 63-72 (2011);incorporated by reference herein). M8 treatment resulted in a ^(˜)5-foldincrease in the protein expression of CD40, and CD86, a ^(˜)3-foldincrease in the expression of CD83, and a ^(˜)2-fold increase inexpression of CD80, compared to cells treated with WT, M5 or poly(I:C).Collectively, these data indicated that M8 was a potent inducer of DCmaturation and activation.

M8 Potentiates Influenza VLP Immunogenicity.

To determine if M8 possessed adjuvant activity in an in vivo model ofvaccination, BALB/c mice were vaccinated by intramuscular injection withVLP co-expressing the HA and NA from H5N1 and HIV GAG protein, incombination with M8, M5 or poly(I:C) (which was previously shown topotentiate responses to influenza antigens (Goff P H et al, PLoS One 8,e79194 (2013); incorporated by reference herein). Three weeks later,mouse sera were collected and analyzed for HAI activity[hemagglutination inhibition assay (HAI) or receptor blocking titers];mice immunized with VLP combined with M8, M5 or poly(I:C) displayed2-3-fold greater HA-specific IgG titers and 2-3 fold higher HAI antibodytiters (p<0.005) (FIG. 24A) compared to mice immunized with VLP alone.M8 was a more potent stimulator of HA-specific IgG and HAI antibodytiters compared to M5 or poly(I:C) (FIG. 24A).

Next, vaccinated mice were challenged with a lethal dose of influenzaH5N1, and lungs were harvested for assessment of influenza replicationand histopathological analysis three days post-infection (FIG. 24B).Immunization with VLP alone resulted in a 4-log decrease in viralplaques, while adjuvantation of VLP with M8, M5 or poly (I:C) resultedin an additional 1-log reduction in viral titer (p<0.05). When comparedto M5-VLP or poly(I:C)-VLP, mice immunized with M8-VLP had the lowestvirus titer in the lungs. These data were further corroborated byhistopathological examination and assessment of lung tissue apoptosis.TUNEL staining revealed that the number of apoptotic cells wasapproximately 3-fold lower in VLP-immunized mice compared to control,and a further 5-10 fold lower when M8, M5 or poly (I:C) were used as anadjuvant (p<0.05, FIG. 24C). Mice immunized with M8-VLP had ^(˜)50%fewer apoptotic cells compared to M5-VLP or poly(I:C)-VLP immunizedmice. Similarly, H&E staining of lung cross-sections indicated completeabsence of edema and inflammation in mice immunized with VLP-adjuvantedwith M8, while non-vaccinated, adjuvant-only vaccinated, and to a lesserextent VLP-vaccinated mice, had signs of inflammation around airways(FIG. 24D). Altogether, these data indicate that VLP combined with M8,M5 or poly(I:C) efficiently blocked influenza virus replication,decreased virus-induced apoptosis, and decreased inflammation in thelungs of vaccinated animals. Of the adjuvants tested, M8-VLP elicitedthe highest level of HAI antibody titers and demonstrated the lowestlevel of influenza-induced lung tissue damage, highlighting the improveddesign and activity of M8.

Antigen Sparing Capacity of M8 in Combination with VLP.

Antigen sparing is regarded as a crucial parameter for pandemic vaccinedevelopment. Because the addition of adjuvant to a vaccine is animportant antigen-sparing strategy (Dormitzer P R et al, Immunol Rev239, 167-177 (2011); incorporated by reference herein), the capacity ofM8 to stimulate immune responses to decreasing doses of VLP wasdetermined. Mice were immunized with 0.5, 1 or 2 μg VLP (as measured byHA content) alone or in combination with M8 (5 μg). Analysis of the HAIantibody titers three weeks showed that 0.5 μg VLP+5 μg M8 resulted in asimilar titer to 2 μg VLP (FIG. 25A). Next, mice were challenged with alethal inoculum of influenza H5N1 (5,000 pfu/mouse). Similar to theresults seen with HAI antibody titers, lung virus titers measured threedays post-challenge were comparable between immunized with VLP alone (2μg) or VLP (0.5 μg) formulated with M8 (FIG. 25B). To determine theminimal dose of M8 that was able to elicit a protective immune response,BALB/c mice were immunized with a single low dose inoculum of VLP (0.5μg), and with varying concentrations of M8 (0.1-5 μg). Three weekspost-immunization, sera were collected to determine antibody titers andmice were challenged with H5N1. Three days after the challenge, lungswere collected to determine virus titers. Addition of as little as 0.5μg M8 resulted in 1.5-fold higher HAI antibody titers compared to VLPalone (FIG. 25C) and greater antibody titers were observed in adose-dependent manner with still higher concentrations of M8. Areciprocal relationship was observed with lung virus titers—higherconcentrations of M8 resulted in lower lung viral loads. The effect wasmaximal in mice immunized with 5 μg M8-VLP (FIG. 25D). Finally, miceimmunized with 0.5-5 μg M8 in combination with VLP all survived the H5N1challenge, although limited weight loss was observed at lower M8concentrations (FIGS. 25E,25F), indicating that M8 (0.5 μg or higher)functioned as an antigen-sparing adjuvant in protecting BALB/c mice froma lethal influenza challenge.

Adjuvant Properties of M8, Alum, AddaVax and Poly(I:C).

The adjuvant activity of M8 was next assessed relative to theFDA-approved adjuvant Alum, AddaVax—an MF59-like adjuvant (Mbow M L etal, Curr Opin Immunol 22, 411-416 (2010); incorporated by referenceherein), and to poly(I:C)—by examining antibody titers, influenza lungtiters, sickness score, weight loss and survival (illustratedschematically in FIG. 26A). Assessment of HA-specific IgG by ELISArevealed ^(˜)2-fold higher antibody levels in mice immunized with M8-VLPcompared to mice immunized with Alum-VLP, AddaVax-VLP or poly(I:C)-VLP(FIG. 26B). Similarly, the HAI assay revealed a ^(˜)1.5-fold higher HAIantibody titer in M8-VLP vaccinated mice (FIG. 26C), compared toAlum-VLP, AddaVax-VLP, or poly(I:C)-VLP. Furthermore, levels ofHA-specific IgM were also higher in M8-VLP immunized animals at fivedays after vaccination (FIG. 26D).

All animals immunized with an adjuvant+VLP formulation survived lethalinfluenza H5N1 challenge (FIG. 27A). Furthermore, animals immunized withVLP in combination with one of the four adjuvants showed no signs(M8-VLP) or temporary slight weight loss after challenge ([LP-Alum,VLP-AddaVax, and VLP-poly(I:C)] (FIG. 27B). In contrast, median survivalfor non-immunized animals or animals immunized with adjuvant-only was9-10.5 days. Animals immunized with VLP had a median survival of 12 days(three animals survived influenza challenge). Post-immunization, thecontrol VLP-immunized and adjuvant-immunized animals all lost weight(FIG. 5B). The health of the animals, as determined by the sicknessscore, was better in animals immunized with M8-VLP, even when comparedto the animals immunized with Alum-VLP, AddaVax-VLP or poly(I:C)-VLP(FIG. 27C). M8-VLP treatment resulted in a ^(˜)1 log-fold lower viraltiters three days post-infection compared to mice immunized withAlum-VLP, AddaVax-VLP, or poly(I:C)-VLP (FIG. 27D). Overall, use of M8as an adjuvant in a formulation with VLP protected mice completely froma lethal influenza challenge and performed better than Alum-VLP,AddaVax-VLP, or poly(I:C)-VLP.

M8 Stimulates Prolonged Immune Responses.

VLP vaccine adjuvanted with M8, Alum, AddaVax or poly(I:C) were testedin order to determine which would best induce a prolonged memoryresponse against influenza challenge. Sera were collected at four weeksand again at four months after immunization. After the second serumcollection, mice were challenged with H5N1 and their weights, survivaland sickness score were assessed (FIG. 28A-28E). At four months afterimmunization, all animals immunized with adjuvanted VLP had detectableantibody titers, although 5-10 fold lower endpoint titers and ^(˜)50%lower in HAI antibody titers were observed in all cases (FIGS. 28A and28B). Mice immunized with VLP adjuvanted with M8, Alum, AddaVax, orpoly(I:C) all survived challenge, with only a 10-15% weight loss (FIGS.28C and 28D); animals also had comparable sickness scores during theweight loss period (FIG. 28E). In contrast, VLP-immunized mice withoutadjuvant had no detectable antibody titers and were not protected fromthe lethal challenge. Altogether, these data indicate that M8, similarlyto Alum, AddaVax and poly(I:C), stimulated a prolonged immune responsecapable of protecting mice against homologous influenza challenge.

M8-VLP Promotes Germinal Center Formation.

Next, the capacity of M8 and other adjuvants to drive germinal center(GC) formation when co-administered with VLP was determined. Initiallymice were immunized IM but no changes were observed with regard to GCformation between different groups and controls. So mice were immunizedby intraperitoneal inoculation with VLP (1 μg, based on HA content),formulated with M8 or poly(I:C) (5 μg), or mixed 1:1 with Alum orAddaVax. Splenocytes from immunized mice were harvested on day 8 andevaluated by flow cytometry ex vivo for the presence ofB220^(hi)CD19^(hi)CD95^(hi)CD38^(lo) GC B cells (FIG. 29A). Compared tomice immunized with VLP alone, a 2.2-fold increase in the frequency ofGC B cells was observed for M8 (p<0.05), 3.5-fold for Alum (p<0.01),4.5-fold for AddaVax (p<0.005), and 1.5-fold for poly(I:C) (FIG. 29B).In addition, the number of IgG1⁺ GC B cells was also determined byintracellular staining. As shown in the bottom panel, the number ofIgG1+ GC B cells was elevated in mice immunized with Alum-VLP (p<0.005)or AddaVax-VLP (p<0.005) compared to VLP alone (FIG. 29C), indicatingthat these two adjuvants induced a T_(H)2-biased response.

M8-VLP Biases CD4+ T Cell Effector Function.

To establish whether M8 induces T_(H)1 or T_(H)2 responses, the levelsof four IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3) were determinedafter immunization using HA coated ELISA plates and respective IgGsecondary antibodies. Immunization of mice with VLP in combination witheither Alum or AddaVax induced higher levels of IgG1, while immunizationwith M8-VLP induced higher levels of IgG2 (FIG. 30A), indicating that M8preferentially induced a T_(H)1-biased response, whereas Alum or AddaVaxpreferentially induce T_(H)2-biased response (Viscaino M L et al, JTransl Med 10, 4 (2012); incorporated by reference herein). To furtherexamine whether M8 promoted T_(H)1/Tc1 cytokine secretion, splenocytesfrom immunized mice were incubated with either HA₅₁₈₋₅₂₆ (10 μg/mL,IYSTVASSL) peptide or VLP (1 μg/mL, based on HA content). B220^(neg)cells were segregated based on surface CD4 and CD8 expression andintracellular staining for IFNγ, IL-2, and TNFα (FIGS. 30B-30E). Invitro re-stimulation of splenocytes with VLP stimulated secretion ofIFNγ in CD8+ T cells (FIG. 30B) and induced the secretion of all threecytokines in CD4+ T cells (FIGS. 30C-30E). The highest induction ofthese three cytokines was observed in splenocytes originating fromM8-VLP immunized animals, followed Alum-VLP, AddaVax-VLP andpoly(I:C)-VLP (p<0.05 between M8-VLP vs. Alum-VLP and p<0.005 betweenM8-VLP vs. AddaVax-VLP for IL-2 and TNFα). These data indicate thatM8-VLP promoted T_(H)1 priming of VLP-stimulated CD4 T cells compared toAlum-VLP, AddaVax-VLP and poly(I:C)-VLP. Additionally, low intracellularlevels of the T_(H)2 cytokine IL-10 were observed in animals vaccinatedwith M8-VLP (FIG. 30F), indicating that M8 does not induce a T_(H)2biased response. Re-stimulation of splenocytes from M8-VLP immunizedmice with HA₅₁₈₋₅₂₆ (IYSTVASSL) peptide did not elicit cytokineproduction by CD8+ T cells, indicating that IP vaccination with M8-VLPdid not promote cross-presentation (Joffre O P et al, Nat Rev Immunol12, 557-569 (2012); incorporated by reference herein).

The invention claimed is:
 1. A synthetic oligoribonucleotide at least 41nucleotides in length that can form a hairpin structure comprising atleast 17 base pairs, the synthetic oligonucleotide further comprising atriphosphate group at a 5′ end of the oligoribonucleotide wherein thesynthetic oligoribonucleotide comprises SEQ ID NO:
 10. 2. The syntheticoligoribonucleotide of claim 1 incorporated into a pharmaceuticallyacceptable carrier.
 3. A pharmaceutical composition comprising asynthetic oligoribonucleotide of claim 1, and a pharmaceuticallyacceptable carrier, and a viral antigen.
 4. The pharmaceuticalcomposition of claim 3 wherein the viral antigen comprises an influenzavirus like particle.
 5. A method of treating a viral infection, themethod comprising administering an effective amount of thepharmaceutical composition of claim 3 to a subject.
 6. An antiviralcomposition comprising a synthetic oligoribonucleotide of claim
 1. 7. Amethod of inducing an antiviral response against a viral infectioncomprising administering to a subject an effective amount of theantiviral composition of claim
 6. 8. The method of claim 7, wherein theviral infection is caused by dengue virus, chikungunya virus, orinfluenza virus.